Centrioles in the cell cycle of L cells

The structure of centrosome in non-synchronous L-cells culture during the cell cycle has been studied. In mitosis, mother and daughter centrioles, which differ in their ultrastructure, are located perpendicularly in the pole of the spindle. Microtubules, meeting in the pole area terminate mainly in electron-dense clottings of fibrillar matter surrounding the diplosoma. In telophase, disjunction of mother and daughter centrioles begins. At the beginning of G1-period, centrioles move off from each other for several micron, and then draw together again without forming diplosome. Pericentriolar satellites form on mother centriole of some cells at this time, they disappear at the beginning of S-period, replication of centrioles begins; daughter centrioles reach the size of mother centrioles in anaphase. During growth and maturation, centrioles in L-cells undergo structural changes similar to those described for SPEV cells (Vorob'ev, Chentsov, 1982). Several types of meeting points for microtubules exist in L-cells during the whole interphase: surface of centrioles per se, pericentriolar satellites, free foci.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Temporal order in mammalian cells. I. The periodic synthesis of lactate dehydrogenase in the cell cycle

Chinese hamster cells were synchronized by the Colcemid-selection system. In cells with a division cycle time of 11.5–12 hr, the activity of the enzyme lactate dehydrogenase (LDH) underwent marked oscillations with a 3.5-hr period. Precipitation of labeled LDH enzyme with specific antibody indicated that the enzyme activity changes were the result of intermittent enzyme synthesis and relatively constant degradation. Inhibition of normal DNA replication with 4 mM of thymidine, while reducing the amount of new enzyme synthesized, did not prevent oscillations from occurring. Similarly, actinomycin D (AcD) added at the time of synchronization allowed some new enzyme synthesis to proceed in an oscillatory manner. LDH synthesis went on at nearly normal rates when AcD was added in the middle of S phase. However, addition of cycloheximide to cultures at any time in the cycle caused an immediate drop in levels of activity and in enzyme protein. The half-life of LDH, calculated either from loss of enzyme activity or precipitable radioactivity in cycloheximide-treated cultures, was between 2 and 2.5 hr.     

Changes in cell distribution during mouse secondary palate closure in vivo and in vitro: I. Epithelial cells

The distribution of epithelial cells around the perimeter of mouse secondary palatal shelves was observed before and after shelf reorientation in vivo and in vitro. Changes in shelf perimeter, cells per micrometer, and cell layering were measured for each of three shelf regions: anterior and posterior presumptive hard and presumptive soft palate at developmental stages which were 30, 24, and 18 hr prior to expected in vivo elevation, after in vivo elevation, and during the course of in vitro elevation. Pronounced increases in numerical cell density and cell layering accompanying shelf reorientation were noted in the superior nasal and mid-oral portions of the shelf perimeter in all three shelf regions with greatest changes noted in the posterior hard palate region. These changes were not attributable to cell division or to perimeter changes. The localized nature of the changes in cell distribution suggest that the underlying mechanisms may also be localized.     

The cell cycle dependence of thermotolerance. I. CHO cells heated at 42 degrees C

We examined the dependence of heat killing and thermotolerance on the position and progression of Chinese hamster ovary (CHO) cells in the cell cycle. We measured cell cycle perturbations and survival of asynchronous and synchronized G1-, S-, and G2-phase cells resulting from continuous heating at 42.0 degrees C for up to 80 hr. Thermotolerance under these conditions was transient in nature, was dependent on the position of cells in the cell cycle, and occurred concurrently with a heat-induced delay of progression of G1- and G2-phase cells. When G1 cells were heated, survival decreased to 25% after 4 hr, at which time the thermotolerance was expressed. For G2 cells survival decreased initially at the same rate (T0 congruent to 3 hr) but thermotolerance was not expressed until approximately 12 hr, at which time the survival was 4%. The rate of decrease in survival was much more rapid for cells heated in mid-S phase (T0 congruent to 0.5 hr), and these cells did not express thermotolerance at a measurable level. Concurrent with the expression of thermotolerance, the progression of cells heated in G1 and G2 was delayed. Following the expression of tolerance, progression resumed at a rate approximately equal to the rate of decrease in survival of the G1 population. Cells heated in mid-S phase continued to progress through the cell cycle until they reached G2, where they were also delayed.     

Centrioles take center stage.

Centrioles are among the most beautiful and mysterious of all cell organelles. Although the ultrastructure of centrioles has been studied in great detail ever since the advent of electron microscopy, these studies raised as many questions as they answered, and for a long time both the function and mode of duplication of centrioles remained controversial. It is now clear that centrioles play an important role in cell division, although cells have backup mechanisms for dividing if centrioles are missing. The recent identification of proteins comprising the different ultrastructural features of centrioles has proven that these are not just figments of the imagination but distinct components of a large and complex protein machine. Finally, genetic and biochemical studies have begun to identify the signals that regulate centriole duplication and coordinate the centriole cycle with the cell cycle.     

The ORC1 cycle in human cells: I. cell cycle-regulated oscillation of human ORC1

Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2-5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring.     

Epithelial bridging of the primary palate: I. Characterization of sub-cultured epithelial cells

Primary palatogenesis involves an intricate array of events. Cell migration, proliferation, differentiation, programmed death, and fusion occur. Prior to fusion, the morphology of the epithelium undergoes marked changes. Epithelial projections form and extend across the fusion site attaching by filopodia to the opposite prominence. By appearance, the epithelium plays a critical role in facial development. In order to monitor epithelial activities, a study was done to isolate and characterize epithelial cells derived from the primary palate. The primary palate was microdissected from day 13 Sprague-Dawley rat embryos, and the epithelium and mesenchyme were separated by enzymatic digestion with a 3% trypsin-pancreatin solution (3:1). All explants were cultured in Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum (FCS), 20 ng/ml epidermal growth factor (EGF), and antibiotics. Explant cells were gathered by trypsin harvesting and sub-cultured. These sub-cultured cells were further characterized. Transmission and scanning electron microscopy showed that the cells retained many morphological features observed in vivo. In passaged cells, type IV collagen, laminin, and cytokeratins were visualized by immunocytochemistry. Gel electrophoresis analysis of the water-insoluble extracts demonstrated major bands of proteins of 50 kD and 44 kD that were synthesized by the epithelial cells but not by the mesenchymal cells. These cytokeratin types are suggestive of a simple undifferentiated embryonic epithelium. The effect of all-trans retinoic acid (RA) on cell number and [3H]-proline incorporation was assessed. At [10(-4)M] and [10(-6)M] retinoic acid resulted in significant inhibition in cell proliferation and amount of proline incorporated, with the greater inhibition occurring in the mesenchymal cells. In the concentrations studied, retinoic acid has an inhibitory effect on the two differently derived cell types. This study established that sub-cultured epithelial cells maintain their phenotype and can be used to study fusion processes. Part 2 will demonstrate how the morphology of the epithelial cells can be modified to produce the changes that are observed during fusion of the primary palate.     

The mechanical basis of cell rearrangement. I. Epithelial morphogenesis during Fundulus epiboly

Many morphogenetic processes are accomplished by coordinated cell rearrangements. These rearrangements are accompanied by substantial shifts in the neighbor relationships between cells. Here we propose a model for studying morphogenesis in epithelial sheets by directed cell neighbor change. Our model describes cell rearrangements by accounting for the balance of forces between neighboring cells within an epithelium. Cell rearrangement and cell shape changes occur when these forces are not in mechanical equilibrium. We will show that cell rearrangement within the epidermal enveloping layer (EVL) of the teleost fish Fundulus during epiboly can be explained solely in terms of the balance of forces generated among constituent epithelial cells. Within a cell, we account for circumferential elastic forces and the force generated by hydrostatic and osmotic pressure. The model treats epithelial cells as two-dimensional polygons where the mechanical forces are applied to the polygonal nodes. A cell node protrudes or contracts when the nodal forces are not in mechanical equilibrium. In an epithelial sheet, adjacent cells share common boundary nodes; in this way, mechanical force is transmitted from cell to cell, mimicking junctional coupling. These junctional nodes can slide, and nodes may appear or disappear, so that the number of polygonal sides is variable. Computer graphics allows us to compare numerical simulations of the model with time-lapse cinemicroscopy of cell rearrangements in the living embryo, and data obtained from fixed and silver stained embryos. By manipulating the mechanical properties of the model cells we can study the conditions necessary to reproduce normal cell behavior during Fundulus epiboly. We find that simple stress relaxation is sufficient to account for cell rearrangements among interior cells of the EVL when they are isotropically contractile. Experimental observations show that the number of EVL marginal cells continuously decreases throughout epiboly. In order for the simulation to reproduce this behavior, cells at the EVL boundary must generate protrusive forces rather than contractile tension forces. Therefore, the simulation results suggest that the mechanical properties of EVL marginal cells at their leading edge must be quite different from EVL interior cells.     

Periplast development in cryptophyceae I. Changes in periplast arrangement throughout the cell cycle

Summary The cell covering (or periplast) of many cryptomonads consists of discrete plate areas precisely arranged over most of the cell periphery. Developmental changes in periplast arrangement that occur throughout the cell cycle are examined here forKomma caudata andProteomonas sulcata [haplomorph]. In both cryptomonads, pole reversal occurs during cytokinesis, necessitating major realignment of the plate areas. Growth of the periplast occurs by addition of new plate areas to specialized regions (termed anamorphic zones) located around the vestibular margins and along the mid-ventral line of cells. Development of the periplast from these regions enables elongation and lateral expansion of cryptomonads throughout cell growth. Observed differences in cell division and periplast development between these genera are closely associated with variations in the arrangement of anamorphic zones.     

Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.