Cell-to-substrate adhesions during spreading and locomotion of carcinoma cells : A study by microcinematography and reflection contrast microscopy

Motility and patterns of adhesion were determined by time-lapse cinematography and reflection contrast microscopy for two types of carcinoma cells, selected for their different motile behavior and not for their malignancy. Cells from the V2 rabbit carcinoma become locomotory soon after having established the necessary contact to the substratum. In contrast, cells from a human epidermoid carcinoma (LICR-OC-1) first attain a fully spread configuration before some cells slightly round up again for a slow locomotory activity of short range and duration. Reflection contrast showed that during spreading and locomotion, the cells from both carcinomas displayed a predominance of grey, the color associated with close contacts. Fully spread cells, on the other hand, presented a multitude of focal contacts in individually different arrangements of black streaks and dots, randomly distributed over the entire cell area. The functional meaning of this heterogeneity in the arrangement of focal contacts in fully spread cells is not yet understood. The importance of close contacts for spreading and locomotion, however, seems to be established and is in agreement with findings reported for other cell types engaged in the same activities. It is therefore suggested that the formation of substrate contacts depends on cellular activity rather than on the cell type.     

Automated analysis of living cells through the quantitative use of automated phase contrast microscopy

A simplified theory of image formation in phase contrast microscopy is presented. It is shown that the phase shift induced in light (related to the refractive index) by the observed object can be reconstructed, point by point, from the phase-contrast digitally sampled image through an appropriate algorithm. This allows one to make quantitative observations on unstained, living cells.     

Automated analysis of living cells through the quantitative use of automated phase contrast microscopy

A simplified theory of image formation in phase contrast microscopy is presented. It is shown that the phase shift induced in light (related to the refractive index) by the observed object can be reconstructed, point by point, from the phase-contrast digitally sampled image through an appropriate algorithm. This allows one to make quantitative observations on unstained, living cells.     

Ribonucleic acid transfer from nucleus to cytoplasm during interphase and mitosis in mouse somatic cells cultured in vitro

Summary Kidney cells from primary cultures of 15-day old mouse embryos were incubated for 2, 5 or 10 min with H³-uridine, then either fixed immediately or incubated again for various periods in a chase medium containing an excess of unlabeled uridine and cytidine. The number of grains over the non-nucleolar part of the nucleus (chromatin), the nucleolus and the cytoplasm were counted on the autoradiograms.The grain count showed that both chromatin and nucleolus incorporate very rapidly H³-uridine from the medium, whereas a time lag elapses before any H³-radioactivity above background is detected in the cytoplasm. Incorporation of H³-uridine into the RNA of the nucleus and the nucleolus is not immediately blocked after chase, suggesting that the labeled precursor pool is not completely washed out from the living cell, or diluted by the excess of unlabeled uridine present in the medium. The grain count over the nucleus and the nucleolus rises for a certain time after chase and then gradually declines; H³-radioactivity appears in the cytoplasm 10 min after chase and keeps rising through a 110-min interval. The experiment, then — even though it suggests that the bulk of cellular RNA is synthesized in the chromatin and the nucleolus and then continuously released into the cytoplasm — does not rule out the possibility that some RNA fraction, characterized by a low turnover rate, is synthesized independently in the cytoplasm.Synthesis of RNA is a continuous process throughout the cell cycle, except during metaphase and anaphase. It ceases at prometaphase after the disappearance of the nucleolus and disintegration of the nuclear membrane, and resumes in early telophase. Part of the chromosomal RNA does not remain associated with the chromosomes through division, but is suddenly released into the cytoplasm when the cell enters metaphase.     

Observations on the cytoplasmic membranes of testicular cells,examined by phase contrast and electron microscopy

In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.     

The process of epithelial cell attachment to glass surfaces studied by reflexion contrast microscopy

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

Probing the dynamics and functions of aurora B kinase in living cells during mitosis and cytokinesis

Aurora B is a protein kinase and a chromosomal passenger protein that undergoes dynamic redistribution during mitosis. We have probed the mechanism that regulates its localization with cells expressing green fluorescent protein (GFP)-tagged wild-type or mutant aurora B. Aurora B was found at centromeres at prophase and persisted until approximately 0.5 min after anaphase onset, when it redistributed to the spindle midzone and became concentrated at the equator along midzone microtubules. Depolymerization of microtubules inhibited the dissociation of aurora B from centromeres at early anaphase and caused the dispersion of aurora B from the spindle midzone at late anaphase; however, centromeric association during prometaphase was unaffected. Inhibition of CDK1 deactivation similarly caused aurora B to remain associated with centromeres during anaphase. In contrast, inhibition of the kinase activity of aurora B appeared to have no effect on its interactions with centromeres or initial relocation onto midzone microtubules. Instead, kinase-inactive aurora B caused abnormal mitosis and deactivation of the spindle checkpoint. In addition, midzone microtubule bundles became destabilized and aurora B dispersed from the equator. Our results suggest that microtubules, CDK1, and the kinase activity each play a distinct role in the dynamics and functions of aurora B in dividing cells.