Non-random Positioning Research Articles

Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae.

The establishment of cell polarity was examined in the budding yeast, S. cerevisiae. The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants. The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells. Later it concentrates at the bud tip in budded cells. At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells. Analysis of unbudded haploid cells has suggested a series of events that occurs during G1. The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus. Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch. By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch. We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus. Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse. Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips. spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein. cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins. Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.

Architectural organization in the interphase nucleus of the protozoan Trypanosoma brucei: location of telomeres and mini-chromosomes.

We studied the spatial organization of chromatin in the interphase G1, S and G2 nucleus of the protozoan Trypanosoma brucei, applying in situ hybridization with conventional fluorescence and confocal scanning optical microscopy. The majority of the trypanosome telomere GGGTTA repeats from different chromosomes were found clustered together, either extending in a network through the nuclear interior or localized at the nuclear periphery. The population of one hundred mini-chromosomes was often asymmetrically located: either clustered in a narrow band in close association with the nuclear envelope or distributed into several clusters that segregated into roughly one half of the nucleus. The nuclear organization may undergo modifications during the cell cycle and development. We conclude that non-random spatial positioning of DNA exists in the nucleus of this protozoan. Finding a high level of structural organization in the interphase nucleus of T.brucei is an important first step towards understanding chromosome structure and functioning and its role in the control of gene expression.

A nucleolar auto-antigen is part of a major chromosomal surface component

Several nucleolar antigens are defined by human autoantibodies. These antigens can therefore be used to follow the fate of nucleolar components through mitosis when this major nuclear structure disintegrates and becomes reassembled in G1-phase. We found that fibrillarin leaves the nucleolus before complete breakdown of this structure and attaches to chromosomes before nuclear envelope breakdown. In mouse, fibrillarin attaches over the chromosomal surface except for the excluded centromeric region. The antigen is transported to the new nucleus via the chromosomes and is last seen on chromosomal surfaces facing the cytoplasm during nuclear envelope reformation. Lamin B reappears on the same chromosomal surfaces before the nucleolar antigen is removed and aggregates for new nucleolar reformation in G1-phase cells. From our observations, we postulate that the antigen acts in concert with other proteins as a nuclear envelope equivalent by forming a protective sheath around the chromosome, that it excludes larger molecules, and helps to separate the chromosomes, in addition to segregation of the ribonucleoprotein (RNP) back to the nucleus for nucleolar reconstruction. We also suggest that the selective retention of these antigens from certain areas on individual chromosomes together with specific lamin B attachment over these chromosomal surfaces allows for a nonrandom positioning of chromosomes in the nucleus.

Chromosome segregation from cell hybrids IV. Movement and position of segregant set chromosomes in early-phase interspecific cell hybrids

We searched for evidence of aberrant movement or position of segregant set chromosomes in C-banded and G-11-banded early-phase hamster-mouse and hamster-human cell hybrids that had been prepared with minimal disruption. No evidence was obtained for an increased frequency of multipolar mitosis, delayed or precocious metaphase congression or anaphase segregation, or for exclusion of chromosomes from the daughter nuclei. However, in hamster-human hybrids, segregant set (human) chromosomes were observed to assume a central position within a ring of hamster chromosomes on the metaphase plate. Such non-random positioning may imply that the centromeres of segregant chromosomes make aberrant, or simply less efficient, attachments to the spindle in hybrid cells. This aberrant position may perhaps result indirectly in chromosome loss by interfering with the normal processes of replication, repair or transcription.

Positioning and stability of mitotic spindle orientation in the neuroepithelial cell

To verify non-random positioning and to define the stability of the mitotic spindle orientation in neuroepithelial cells of mouse foetuses, computer — assisted morphometric analysis at the light microscopy level was performed. It was confirmed that the mitotic spindle axis is positioned non-randomly in relation to the cell polarity axis and could be displaced only within a narrow range. This orientation was found to be attained at metaphase and it does not change until telophase is completed. However, in relation to the long axis of the neural tube the mitotic spindle axis was found to be positioned randomly. In the light of these findings centrosome movement and positioning are discussed.

Nonrandom positioning of Golgi apparatus in pancreatic B cells.

The Golgi apparatus has a polarized distribution in a variety of cell types, and in certain epithelia its intracellular position correlates with the direction of vectorial transport and release of secretory products. We have studied quantitatively the orientation of the Golgi apparatus in pancreatic islet B cells by examining its location with respect to the juxtacapillary face of the cell. This was done a) at the light microscope level on semithin sections of osmium-impregnated islets, and b) at the ultrastructural level on thin sections of intact pancreas. The data obtained with both methods show that in pancreatic B cells the Golgi apparatus is preferentially located in the position opposite to the juxtacapillary face of the cell. These results suggest a structural polarization of B cells, which may be important for the coordinate function of islets of Langerhans.

The structure and position of mouse centromeric heterochromatin in BrdU-labelled chromosomes and diplochromosomes stained by the pH 10.4 method.

A mouse cell line of C57Bl/6J spontaneous melanoma (clone PG 19), and a C-type virus transformed cell line (G-8 clone 124) originating from normal Balb/c mice were used in a study of the centromeric heterochromatin region of BrdU-labelled chromosomes stained by the Giemsa pH 10.4 method. Three possible explanations for the generation of compound lateral asymmetry within the centromeric heterochromatin region of the laboratory mouse are discussed: 1) inverted translocation; 2) centric fusion followed by paracentromeric fission and 3) inversion of part of the centromeric satellite DNA. These processes could be of considerable genetic and evolutionary significance. The non-random spatial position of unstained and dark stained C-bands in BrdU-labelled diplochromosomes of endoreduplicated cells can be explained as being due to the localization of the old and new DNA chains in a unineme chromatid model. The late replicating regions are shown to be located on the inside of the half-chromatid close to the axial symmetry axis of the metaphase chromosome.

Non-random position of the A-T rich DNA sequences in early embryos of Drosophila virilis.

Examination of early embryos of Drosophila virilis by light and electron microscopy has shown that the A-T rich satellite DNA sequences have a non-random distribution within the nuclei. As observed by 33258 Hoechst staining and fluorescent microscopy, these sequences are consistently found to be located on the sides of the nuclei nearest to the vitelline membrane. This arrangement of the A-T rich sequences has been observed from the syncytial balstoderm stage into the gastrula stage where, in each nucleus, the satellite DNA sequences remain at a point nearest the topological outside of the organism.

Adult male positioning in baboon progressions: order and chaos revisited.

Evidence of nonrandom positioning among adult males is crucial for a protection theory of the spatial organization of baboon progressions. In a recent study it was suggested that systematic positioning of troop members other than mothers and infants is so slight and rare that progressions may be regarded as essentially random. This suggestion depends upon debatable methodological points presumably downgrading previous findings of nonrandom order. Reanalysis of data from this study revealed numerous analytical and statistical problems, as well as serious calculation and other errors, and showed that the findings are consistent with results of the present and previous research. Adult males tended toward the front or back of progressions, a tendency which was intensified in potentially dangerous situations. Dominant males were disproportionately more often frontward and subordinate males rearward. Nonrandom order, which was found for a variety of circumstances at high levels of statistical significance, was unusually general in that it occurred in 6 studies, 7 troops, 2 species, and 5 locations. Such generality is consistent with a protection theory postulating phylogenetic underpinnings of a sociospatial organization which allows an advanced primate to adapt to terrestrial coexistence with predators.

Remote Sensing for the Determination of the Woody-to-total Area Ratios in Mixedwood Forest Stands

Leaf area index (LAI) is a biophysical variable that is important for estimating processes such as productivity and yield rates in forest stands. Indirect methods for estimating LAI (e.g., digital hemispherical photography (DHP)), provide efficient and reliable measurements. However, DHP utilizes gap-fraction-based methods that are unable to both discriminate between leafy and other plant material and compensate for the non-random positioning of canopy elements. Therefore, LAI estimates obtained with these techniques can be termed effective leaf area index (LAIe). Woody-to-total area ratios are applied to these measurements to compensate for the inclusion of non-photosynthetic canopy components, but these parameters vary amongst tree species and are not well studied. This project attempts to determine LAI for mixedwood stands (i.e., deciduous and coniferous) by examining the woody-to-total area ratio for species in the Haliburton Forest located in southeastern Ontario. Two sets of DHPs were taken for 23 stem-mapped forest plots during field campaigns carried out in leaf-on and leaf-off periods to establish the impact of non-photosynthetic materials on estimating LAI. For solely deciduous stands, the woody-to-total area ratio is assumed to be directly equal to the difference in LAIe values between these two periods. For purely coniferous stands, the woody-total area ratio will be derived in the same manner for leaf-on and leaf-off conditions (i.e., with correction factors applied based on published ratios and corresponding to the basal area ratio of conifers). For mixed stands, the proportions of conifers (in terms of BA) to deciduous species will be used to determine appropriate models for estimating LAIe.