Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.     

Microviscosity changes during differentiation of neuroblastoma cells

Microviscosity $\text{η}$ of the plasma-membrane lipid matrix was measured in exponentially growing and differentiating C1300 mouse neuroblastoma cells, attached to a glass substratum, by fluorescence polarisation of 1,6-diphenyl-1,3,5-hexatriene. Upon differentiation $\text{η}$ decreases progressively, reaching values below those observed in the growth phase. Treatment of the cells with dipalmitoyl phosphatidylcholine vesicles reversibly inhibits morphological differentiation. The results show that a high membrane fluidity is a prerequisite for differentiation.     

Multiple sites for the initiation of microtubule assembly in mammalian cells.

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.     

Influence of epidermal growth factor and cyclic AMP on growth and differentiation of palatal epithelial cells in culture

A serum-free, hormonally defined medium was developed which supports growth and differentiation in primary culture of epithelial cells from prefusion embryonic mouse palatal shelves. Using this culture system, medial epithelial programmed cell death was investigated. In the absence of EGF, medial epithelial cells undergo cell death and detach from the substratum by 24 hr of culture. The addition of EGF alone or in combination with various agents which increase intracellular cyclic AMP levels prevented medial epithelial cell death in both cell and organ culture. EGF appeared to exert its most dramatic effect in cell culture on growth and differentiation of the squamous oral epithelial cells. In addition, EGF and agents such as 8-bromo-cyclic AMP, dibutyryl cyclic AMP, or cholera toxin synergistically stimulated the appearance of a long-lived, rapidly proliferating cell type by Day 4 of culture. Our results suggest that both EGF and cyclic AMP together may be important in regulating proliferation of embryonic palatal epithelial cells.     

Regulation of the cytoskeleton in mesothelial cells: Reversible loss of keratin and increase in vimentin during rapid growth in culture

Human mesothelial cells grew rapidly in culture when provided with serum, EGF, and hydrocortisone, adopting a fibroblastoid shape and forming parallel, multilayered arrays at saturation density. In the absence of EGF, the cells grew slowly to a flat, epithelioid monolayer similar to their normal pattern in vivo. Mesothelial cells normally have a high keratin and a low vimentin content in vivo. In culture, rapidly growing cells greatly reduced synthesis and content of their four major keratins to levels undetectable by immunofluorescence in most cells, but keratin synthesis and content returned to high levels whenever growth slowed. Vimentin synthesis and content was high during serial culture, but decreased several-fold in nondividing cells. The unique ability of the mesothelial cell to reversibly alter its morphology and intermediate filament composition is of unknown function and mechanism, but accounts for the morphological heterogeneity and the presence of keratin-negative cells in mesotheliomas.     

Heparan sulfate proteoglycans of human neuroblastoma cells: Affinity fractionation on columns of platelet factor-4+

Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca²⁺ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C₁₈ cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of “singlechain” heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.     

Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties

The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.     

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.     

Mouse monoclonal antibodies can nonspecifically inhibit the proliferation of human T-cell growth factor-dependent T cells

A large series of mouse monoclonal antibodies was found to inhibit the proliferation of T-cell growth factor (TCGF)-dependent human T-cell blasts as measured by the incorporation of tritiated thymidine. The specificity of the antibody appeared to be irrelevant for inhibition and two T-cell-specific antibodies did not prevent the absorption of TCGF by treated T cells. It is suggested that the antibodies function by the indirect release of suppressor factors by Fc receptor-bearing TCGF-dependent cells.     

Inhibition by neuroblastoma growth inhibitory factor of ascites-type neuroblastoma cell growth in coculture with normal glioblasts

A new ascites type neuroblastoma clone (NAs-1), which is characteristic both in anchorage-independent growth and catecholaminergic functions, attached on the monolayer culture of glioblasts and was subjected to morphological differentiation including the extrusion of neuronal processes. Other conventional neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) as well as NAs-1 in cocultured with normal glioblasts underwent a decrease in cell growth rates and DNA synthesis under the effect of the neuroblastoma growth inhibitory factor (NGIF) produced by glioblasts. After their NGIF production had been reduced by u.v. irradiation, glioblasts lost the growth-inhibitory and differentiation-promoting effects in coculture with NAs-1. The supplement of NGIF into u.v.-treated glioblasts restored the dose-dependent growth inhibition of NAs-1. The addition of nerve growth factor into the coculture system brought about neither the marked effect on growth inhibition of NAs-1 nor the morphological differentiation. The results imply a direct function of NGIF on the paracrine regulation of neuroblastoma cell growth in the coculture with normal glioblasts.     

Analysis of early events occurring during neonatal induction of tolerance to histoincompatible cells in mice

Mice have been analyzed at different times post neonatal inoculation of F₁ hybrid lymphoid cells for their ability to respond to foreign MHC antigens expressed on the tolerizing cell population, and to impart that response phenotype to normal adult spleen cells. At early times following neonatal challenge with F₁ spleen cells all mice produced serum immunoglobulins of F₁ donor origin which inhibited the response of normal adult cells. The antigenic specificity of the inhibitory factors suggests a role for an antireceptor antibody in the inhibition seen, and the presence of such serum-mediated inhibition is indicative of (and may be causally related to) the late appearance of a cell-mediated suppressor mechanism in these mice.     

Timing the early events during sea urchin fertilization

To determine precisely the timing, duration, and sequences of the earliest events during sea urchin (Lytechinus variegatus) fertilization, the bioelectric recordings of microelectrode-impaled eggs were electronically superimposed, by video mixing, over the microscopic differential interference contrast image of the same egg at insemination. Videotape analysis, utilizing a slow-motion analyzer, demonstrates that the successful sperm triggers the bioelectric membrane potential reversal within 3.36 +/- 3.02 sec (0.72-9.76 sec range; sigma = 23 eggs) of sperm-egg attachment. This sperm, actively gyrating about its attachment site, is indistinguishable from the other, unsuccessful sperm until 12.66 +/- 2.72 sec (6.72-16.60 sec range; sigma = 15) later when the sperm tail ceases its beating and sperm incorporation ensues. The cortical granules begin to discharge, and the fertilization coat starts to elevate at the fusion site at 20.79 +/- 3.18 sec (13.62-26.08 sec range; sigma = 12) after the onset of the fertilization potential, i.e., an average of about 8 sec after the cessation of sperm-tail motility during incorporation. In most cases, the bioelectric responses starts within 7 sec of sperm adhesions; if the data are analyzed excluding the few slow cases, the fertilization potential is found to start 1.93 sec (+/- 1.28 sec) after sperm attachment. These results indicate that the first successful sperm triggers the fast block to polyspermy within 3.4 sec, perhaps as quickly as 1.9 sec, of sperm-egg adhesion, about 13 sec before the first morphological indication of fertilization, and about 21 sec before the characteristic elevation of the fertilization coat responsible for the late block to polyspermy.     

Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxococcus xanthus

Myxococcus xanthus is a bacterium that forms multicellular fruiting bodies in response to starvation. The initiation of fruiting body formation is cell density dependent, and we suggest that cells measure their cell density by titering the extracellular concentration of excreted adenosine. Our evidence is as follows: (1) At low cell densities fruiting body formation does not occur unless adenosine is added. (2) Norit, a substance that binds purines, inhibits fruiting body formation, and this inhibition is reversed by adenosine. (3) Cells labeled with [¹⁴C]carbonate excrete [¹⁴C]adenosine which is bound by the Norit. Furthermore, [¹⁴C]adenosine is excreted by developing cells at a concentration that will induce fruiting body formation at low cell density. The extracellular adenosine concentration increases with the cell density over a broad range of densities. (4) Hadacidin, an inhibitor of de novo AMP synthesis, inhibits fruiting body formation, and inhibition by hadacidin can be reversed with adenosine. Adenosine also appears to be involved in the aggregation process because the shape and size of the fruiting bodies are sensitive to the external concentration of adenosine.     

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.     

Rapid electroelution of nucleic acids from agarose and acrylamide gels

The alkaline/filter DNA elution technique measures single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [³H]thymidine-labeled DNA of γ-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of “test” DNA (i.e., ¹⁴C-labeled DNA) relative to the elution of “reference” DNA (i.e., ³H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of “test” DNA cluted relative to “reference” DNA from unincubated cells. For Chinese hamster V79–171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24°C) of ～0.190 and ～0.017 min^?1.     

Studies of the kinetics and ionic requirements for the phosphorylation of ribosomal protein S6 after fertilization of Arbacia punctulata eggs

Fertilization of the eggs of the sea urchin Arbacia punctulata is followed by the phosphorylation of ribosomal protein S6. The increase in phosphorylation starts at the same time that protein synthesis begins to increase, and leads to the appearance of mono-, di-, and triphosphorylated S6 derivatives. Essentially all the S6 is phosphorylated by first cleavage. This phosphorylation requires the occurrence of both the normal Ca²⁺ transient and the consequent Na⁺H⁺ exchange. Protein synthesis can be partially activated by an increase in intracellular pH brought about by weak bases, but this neither causes S6 phosphorylation, nor the inactivation of the specific S6 phosphatase present in unfertilized Arbacia eggs.     

Erasure of the memory response in MEL cells

When MEL cells are reexposed to DMSO after an interruption in inducer treatment, they can initiate commitment to differentiation without the lag period observed after the primary exposure to inducer. This property is known as memory. Here we have employed metabolic inhibitors to analyze the basis of the memory response. Treatment of cells with cycloheximide or cordycepin during the inducer withdrawal period causes memory erasure. Cells must recapitulate an entire lag period upon reexposure to DMSO. The memory response is maintained, however, if cells are treated with metabolic inhibitors in the presence of DMSO. Our results suggest that the capacity of MEL cells for memory requires the synthesis of cell components which are normally stable in the absence of DMSO. Experiments involving reciprocal shifts between two different inhibitors have been performed. Evidence is presented that the process leading to the initiation of commitment is composed of at least three components acting in sequence.