Chromosomal Fibers Research Articles

Biological Observation with Laser Plasma X-Rays.

Biological observation with soft X-ray microscopy using intense laser-produced plasma X-rays is reviewed. When laser is focused on a solid target at the intensity of 1012-1015W/cm2, plasmas are produced on the solid surface and emit intense soft X-rays. The X-rays are useful for soft X-ray microscopy which draws much attention recently. Soft X-ray microscopy is developed extensively using synchrotron radiation and applied to biological observation. However, for the observation of hydrated biological specimens, synchrotron radiation is not intense enough to circumvent image blurring caused by radiation damage and thermal diffusion. The single shot exposure of laser-produced plasma X-rays will have enough intensity for imaging hydrated biological specimens and will circumvent these problems. Images of hydrated human chromosome fibers obtained with soft X-ray contact microscopy using laser-produced plasma X-rays are shown at the resolution of-10 nm as an example. The results indicate a big potential of laser-produced plasma X-rays for soft X-ray microscopy of hydrated biological specimens.

Morphological aspects of chromosome spindle fibres inMesostoma: ?microtubular fir-tree? structures and microtubule association with kinetochores and chromatin

Bipolarly oriented bivalents in spermatocytes of the turbellarianMesostoma ehrenbergii, displaying fast oscillatory movements in metaphase, were studied with the electron microscope. Kinetochores and chromosome fibres were reconstructed using serial sections cut perpendicular to the spindle axis. Only a small proportion of kinetochore microtubules (kMT) is continuous between the kinetochore and the centrosome. kMTs intermingle with non-kinetochore microtubules (non-kMTs), partly inclined with regard to the kMTs, thus forming a chromosome fibre MT lattice. This resembles the “microtubular fir-tree” structures (MTFT) described by Bajer and Mole-Bajer inHaemanthus endosperm mitosis. A minimal function of the MTFT may be the anchorage of kMTs in the polar region. Regarding the association of MTs with the chromosome, three types of attachment can be discriminated: (1) “normal” insertion of kMT plus ends in the kinetochore, (2) penetration of kinetochores and deep insertion in the chromatin, and (3) lateral attachment with kinetochore and chromatin. Lateral association of MTs seems to be mediated by filamentous crossbridges. The observations are discussed in connection with possible behaviour of kMTs during kinetochore movement.

Chromosome fiber dynamics and congression oscillations in metaphase PtK2 Cells at 23°C

A bioriented chromosome is tethered to opposite spindle poles during congression by bundles of kinetochore microtubules (kMts). At room temperature, kinetochore fibers are a dominant component of mitotic spindles of PtK2 cells. PtK2 cells at room temperature were injected with purified tubulin covalently bound to DTAF and congression movements of individual chromosomes were recorded in time lapse. Congression movements of bioriented chromosomes between the poles occur over distances of 4.5 microns or greater. DTAF-tubulin injection had no effect on either the velocity or extent of these movements. Other cells were lysed, fixed, and the location of DTAF-tubulin incorporation was detected from digitally processed images of indirect immunofluorescence of an antibody to DTAF. Microtubules were labeled with an anti-beta tubulin antibody. At 2-5 minutes after injection, concentrated DTAF-tubulin staining was seen in the kinetochore fibers proximal to the kinetochores; a low concentration of DTAF-tubulin staining occurred at various sites through the remaining length of the fibers toward the pole. Kinetochore fibers in the same cell displayed different lengths (0.2 to 4 microns) of concentrated DTAF-tubulin incorporation proximal to the kinetochore, as did sister kinetochore fibers. Ten minutes after injection, the lengths of DTAF-containing chromosomal fibers were greater than expected if incorporation resulted solely from the lengthening of kinetochore microtubules due to congression movements of the chromosomes. Besides incorporation as a result of chromosome movement, two other mechanisms might explain the length of the DTAF-containing segments: 1) a poleward flux of tubulin subunits (Mitchison, 1989) or 2) capture of DTAF-containing nonkinetochore microtubules.

Quantitative Energy-Filtered CRYO-EM of Hydrated Chromosome Fibers, Viruses, and Heavy Atoms

We have calculated and experimentally measured the extent to which energy filtration improves the S/N in phase- and scattering-contrast images of frozen-hydrated molecules. Multiple scattering calculations based on PW (partial wave) complex scattering amplitudes agree quantitatively with 80 KV image intensities of 50-500nm-thick ice. For an objective aperture of 2.5 nm-1 the PW elastic mean free path is 280 nm. From the ratio of filtered to unfiltered image intensities from 50-500 nm of ice at 80 KV, we calculate an empirical inelastic mean free path of 180 nm. The PW calculations show that Beer's Law should be obeyed for thicknesses up to five mean free paths, whether or not energy filtration is used. Thus the bright-field signal can be used to determine the absolute mass distribution of molecules embedded in thick ice layers.Energy filtration removes background intensity due to inelastic scattering (Fig.1a), and increases the scattering signal from molecules (becoming the sum of both elastic electrons excluded by the objective aperture and inelastic electrons excluded by the energy filter). As the result, the calculated contrast is 2-15 times greater in the Zeiss EM902 than in a TEM without energy filtration (Fig.1b). 80 KeV energy-filtered images of frozen-hydrated molecules should have a higher S/N than unfiltered images taken at intermediate or high voltage (Fig. 1c,d). TMV contrast and S/N is improved by filtration and agrees with calculations (Fig.1b,2). The advantages of energy-filtration are important to the microscopy of chromatin fibers, which are excluded from ice layers ≤200 nm and viruses, which are usually excluded from layers ≤100 nm. Radial density reconstructions from near-focus energy-filtered images of frozen-hydrated chromatin from Thyone sperm show that the centers are not hollow, but very dense, probably containing DNA (Fig.3). Energy-filtered images of icosahedral viruses in even the thinnest possible ice have a much improved S/N ratio, decreasing the dose required for analysis (Fig.2a,b).

Fine structure of unstained human chromosome fibres dried with no fixative as observed by X‐ray contact microscopy

X-ray contact microscopy was used to examine unstained human chromosomes dried without any fixative. This is the first report of the observation of single human chromosome fibres at high resolution without any modification(s) such as staining or fixation. The 'beads-on-a-string' structure was observed in stretched portions of chromosome fibres. The diameters of the thin filaments and small particles were 7-15 nm (mean +/- SD of 12.2 +/- 2.1 nm) and 16-69 nm (31.4 +/- 13.2 nm), respectively. These sizes correspond to those of fibres composed of nucleosomes (10-15 nm in diameter) and particle units (15-50 nm in diameter) called superbeads (unit structures composed of a multiple association of nucleosomes). These results are in good agreement with the current nucleosome and superbead model for chromosome fibre structure.

Application of X-ray microscopy to biology.

The progress in the application of X-ray microscopy to biology is reviewed with respect to the observation of human chromosomes. Isolated chromosomes, unstained and dried without fixative, were observed by X-ray contact microscopy at high resolution. The 'beadson- a-string' structure was observed in stretched portions of chromosome fibers. Detailed analysis of the results shows that the diameters of the thin filaments and small particles were 12.2± 2.1 nm and 31.4±13.2 nm, respectively. These sizes correspond to those of fibers composed of nucleosomes and particle units called superbeads revealed by electron microscopy. Progress in efforts toward the observation of hydrated specimens and three-dimensional observation is also reviewed. The problem of radiation damage tospecimens caused by imaging X-rays may become serious for the application of X-ray microscopy to hydrated intact biological organisms, but may be circumvented by making a proper choice of X-ray source and optics.

Identifying the site of microtubule polymerization during regrowth of UV-sheared kinetochore fibres using antibodies against acetylated alpha-tubulin

Areas of reduced birefringence (ARBs) produced on chromosomal fibres of crane-fly spermatocyte spindles by ultraviolet microbeam irradiation move poleward. The ARB is due to the depolymerization of the microtubules in that area, and its poleward motion is due in part to the lengthening of that part of the kinetochore fibre which is left attached to the kinetochore after shearing the microtubules. We tested whether the lengthening of this fibre is due to the polymerization of microtubules at the growing edge of the ARB by staining growing fibres in irradiated spindles with antibodies to tubulin and to acetylated tubulin. We have previously argued that newly-polymerized kinetochore microtubules are not acetylated, whereas older kinetochore microtubules are (Wilson & Forer, 1989). Therefore we expected to see an absence of staining with antibodies to acetylated tubulin at the edge of the ARB if microtubules were polymerizing there. There is no absence of staining, however, which suggests that growth of the sheared microtubules does not occur at the ARB edge. Other possibilities are discussed.

Rapid kinetochore movements in Mesostoma ehrenbergii spermatocytes: Action of antagonistic chromosome fibres

AbstractChromosome movements in Mesostoma ehrenbergii spermatocytes were studied using conventional video light microscopy. Kinetochore regions of the three bipolarly oriented bivalents displayed periodic back and forth movements directed to both poles at metaphase I, leading to periodic lenght changes of the bivalents. Velocity was 8–10 μm/min (maximum 17 μ/min), about one order of magnitude higher than the normal meiotic or mitotic chromosome movements of other species. One cycle of movement lasted for about 100 seconds. The movement of kinetochore regions implies that the antagonistic chromosome fibres periodically grow (assemble) and shorten (disassemble) at comparable rates. Poleward movements must be caused by forces generated in disassembling fibres, whereas movements away from the poles, accompanied by fibre growth, are probably brought about by the internal elastic force of the chromosomes. Antagonistic fibres of a bivalent can operate in or out of phase. The movements of the three kinetochore regions are coordinated insofar as growing and shortening fibres coexist in a half‐spindle at almost any time [Fuge, H. (1987): European Journal of Cell Biology 44:294–298]. These observations are discussed in terms of microtubule dynamics.

Microtubule dynamics in the chromosomal spindle fiber: Analysis by fluorescence and high‐resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.

Quantitative Diameter Measurements of Chromatin and TMV Fibers in the Freeze-Dried State

Although there is general agreement that Inactive chromosome fibers consist of helically packed nucleosomes, the pattern of packing is still undetermined. Only one of the proposed models, the crossed-linker model, predicts a variable diameter dependent on the length of DNA between nucleosomes. Measurements of the fiber diameter of negatively-stained and frozen- hydrated- chromatin from Thyone sperm (87bp linker) and Necturus erythrocytes (48bp linker) have been previously reported from this laboratory. We now introduce a more reliable method of measuring the diameters of electron images of fibrous objects. The procedure uses a modified version of the computer program TOTAL, which takes a two-dimensional projection of the fiber density (represented by the micrograph itself) and projects it down the fiber axis onto one dimension. We illustrate this method using high contrast, in-focus STEM images of TMV and chromatin from Thyone and Necturus. The measured diameters are in quantitative agreement with the expected values for the crossed-linker model for chromatin structure

Cryo-electron microscopy of chromosome fibers is consistent with the crossed-linker model for chromatin structure

Although there is general agreement that inactive chromosome fibers consist of helically packed nucleosomes, the pattern of packing is still disputed. The nucleosome itself is composed of a highly conserved “core” comprised of 146bp of DNA and a histone octamer, and a variable “linker” comprised of 20-100bp of DNA bound to histone H1. The models of chromatin structure can be distinguished by their dependence upon linker length. The solenoid model for chromatin structure is a single helix with constant pitch (11 nm) and constant diameter (25-30nm). The twisted-ribbon model is a double helix with variable pitch (26-40nm) and constant diameter (30nm). The crossed-linker model is a double helix with conserved pitch (26-28nm) and variable diameter (26-40nm). Measurements of the diameter and apparent helical parameters of negatively-stained chromatin from Thyone sperm (87bp linker) and Necturus erythrocytes (48bp linker) are in agreement with a left-handed crossed-linker model, and in disagreement with the solenoid and twisted-ribbon models.

Arrangement and dynamics of spindle structures in crane fly spermatocytes seen with video-enhanced contrast differential interference contrast microscopy

This is a report on the arrangement and dynamic behaviour of microtubule fibres investigated by video-intensified microscopy (Allen Video Enhanced Contrast —Differential Interference Contrast, AVEC-DIC) in spindles of living crane fly spermatocytes. The spindle was found to contain numerous fibrils, each fibril probably consisting of several microtubules. The fibrils are oriented between the poles and, due to slight inclinations, towards each other, frequently form arrowhead-like structures or points of intersection. This leads to an overall lattice-like arrangement. The fibrils display flickering motions visible in real time recordings. The observations are discussed in relation to ultrastructural data on chromosome fibre architecture from previous studies.

Action of teniposide on the ultrastructure of human lymphocytes chromosomes.

Action of Teniposide on the Ultrastructural Aspect of Human Metaphasic Lymphocytes ChromosomesTeniposide tested “in vitro” on human lymphocytes cultures of 24 hours, at various concentrations, inhibits the formation of the mitotic metaphase plates and brings about thickening of the chromosomal fiber, observed in electron microscopy.After 3 hours of exposition in the culture, teniposide induces numerous chromosomal aberrations: gaps, breaks, exchanges.

Relationship between the arrangement of microtubules and chromosome behaviour of syntelic autosomal univalents during prometaphase in crane fly spermatocytes.

During meiotic prometaphase of crane fly spermatocytes, syntelic autosomal univalents are able to move between the spindle poles several times. The relationship between the behaviour of chromosomes and the arrangement of microtubules during this stage was studied using a fixation technique (Nicklas et al. 1979) which makes it possible to examine a certain cell in an electron microscope after live observation. After reorientation, when a syntelic univalent had started moving towards the opposite spindle pole, a short chromosome fibre was found. When a univalent had reached the equator, a chromosome fibre could be traced up to the spindle apex. During the movement towards the opposite spindle pole the degree of disorder in the chromosome fibre was high, whereas it was low in the fibre of a motionless univalent. The degree of disorder was determined by the relative proportion of skew fibre microtubules. At the beginning of reorientation a chromosome fibre was still present, but later, it was no longer possible to recognize such a fibre. Instead of a chromosome fibre, a bundle of microtubules laterally associated with the kinetochore was observed. Some microtubules of this bundle had a direct contact with the kinetochore. These observations strongly hint that the laterally associated microtubules have an important function in the reorientation of syntelic autosomal univalents.

The distribution of intermicrotubular bridges in meiotic spindles of the crane fly.

The distribution of intermicrotubular bridges in spindles of tipulid spermatocytes (Pales ferruginea, first meiotic division) was analyzed using serial sections of pre-selected cells. Bridges were found in all spindle regions, including kinetochore microtubules and free microtubules in the chromosome fiber. The dimensions of bridges were variable, ranging between 60 and 300 A in length and 40 and 190 A in thickness. Bridges seem to be randomly distributed. No accumulation in or absence from particular spindle regions was detected. Quantitative analysis revealed a linear, positive correlation between the number of microtubules and the number of microtubule pairs capable of forming bridges and, on the other hand, between microtubule pairs and intermicrotubular bridges. The possible composition and significance of bridges are discussed.

Three-dimensional architecture of chromosome fibres in the crane fly: co-oriented autosomal bivalents and amphitelic sex univalents during prometaphase

A technique for the fixation of cells during live observation (Nicklas et al. 1979) was used to investigate chromosomes which were moving at the time of fixation. Chromosome fibres were reconstructed by tracking their microtubules in longitudinal serial sections. A considerable proportion of non-kinetochoric microtubules (free microtubules, fMTs) is skewed with respect to the fibre axis. These skew fMTs contribute to the degree of disorder. It was found that the difference in the relative proportion of skew fMTs between “active” fibres (oriented in the direction of movement) and “passive” fibres (oriented backwards) is significantly correlated with the chromosome velocity (correlation coefficient r=0.796, P=0.01). It can be concluded that the pulling force generated in the chromosome fibre is a function of skew fMTs.

An immunofluorescent study of the microtubule organization in Trichomonas vaginalis using antitubulin antibodies.

The flagellated protozoon Trichomonas vaginalis, parasite of the human urogenital tract, possesses a well developed microtubule system organized in highly differentiated structures. We have shown by immunoblotting that monospecific anti-sheep brain tubulin antibodies are able to react with the microtubular tubulin of T. vaginalis. These antibodies were used to study the microtubular system of T. vaginalis both in interphase and mitosis by indirect immunofluorescence. The interphase microtubular pattern, characterized by an axostyle, a pelta, four anterior flagella, and a recurrent flagellum, displayed remarkable changes at the onset of mitosis: the axostyle disappeared, and two pole bodies connected by a short spindle became evident; chromosomal fibers arose while pole-to-pole fibers elongated. The last phases of mitosis were marked by the disappearance of chromosomal fibers, the appearance of two small axostyles, and the depolymerization of the pole-to-pole bundle. At the end of mitosis, the normal interphase microtubule pattern was observed.

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.

Elementary structures in polytene chromosomes of Drosophila melanogaster

Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (26–27 chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 A in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.

A model for chromosome movement based on lateral interaction of spindle microtubules

Quantitative morphological studies of meiotic spindles in the crane fly Pales ferruginea (Fuge, 1980, 1984, 1985) were the basis for the development of a model explaining anaphase chromosome transport in higher eukaryotes. Two main features of chromosome fibres were important for the model: (1) the existence of microtubules oriented obliquely with respect to kinetochore microtubules, and (2) a higher degree of disorder in fibres exerting a pulling force. It is postulated that microtubules of the same polarity being inclined to each other at a certain angle are able to slide past each other by means of mechano-chemically active side-arms working in alternate succession. Sliding is suggested to lead to a displacement of microtubules and chromosomes in direction towards the poles. Furthermore, it is suggested that the chromosome fibre in anaphase becomes progressively disintegrated by fragmentation and disassembly of microtubules. Fragmentation may be induced by bending stress within the dynamic system.