Role of the cytoskeleton in the spreading process of epithelial cells

The role of microtubules and actin filaments in spreading of the IAR-2 cells isolated from the rat liver was studied. At the glass surface in the standard medium the cells rapidly assumed a discoidal form soon after inoculation. In the colcemid-containing medium the spreading is disturbed and delayed. In the cytochalasin D-containing medium the cells form two or more long processes. The effects of these drugs are reversible. It is supposed that microtubules are essential for sending cytoplasmic processes and stabilizing those processes and lamellae which have no numerous and stable contacts with the substrate, e.g., the processes which form at the early stages of spreading or the elongated processes of polarized cells. Bundles of actin microfilaments are essential, in particular, to ensure the discoidal form of epithelial cells. Microtubules appear to prevent the actin cytoskeleton contraction.     

Role of feeder cells in spreading and cytoskeleton organization of newborn rat keratinocytes.

We studied the effect of feeder cells (fibroblasts) and a mixture of the extracellular matrix components, Matrigel, on spreading and cytoskeleton organization of newborn rat keratinocytes (REK). REK formed lamellipodia on being plated together with feeder cells and on the Matrigel as a substrate whereas the same REK plated alone on a plastic surface formed filopodia. REK lamellipodia formation in co-cultures depended on the fibroblast addition time. Although conditioned medium from fibroblast cultures was not enough to induce lamellipodia, the extracellular matrix left after fibroblast removal was as effective as Matrigel. Our results indicate that lamellipodia formation seems to depend on the factor(s) secreted by fibroblasts and associated with the extracellular matrix.     

Role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells

The in vivo site of influenza virus infection is a polarized epithelium, and it is well established that the virus preferentially enters from the apical surface of polarized epithelial cells; however, many of the molecular events involved during the endocytosis of the virus into polarized epithelia remain unclear. Here we examined the role of actin microfilaments and the myosin VI motor protein during influenza entry into a panel of polarized and non-polarized cells. By treatment of cells with cytochalasin D and jasplakinolide, we show that influenza virus entry into all the polarized epithelial cells tested requires actin dynamics, with a specific role for the actin cytoskeleton in the process of virus internalization from the plasma membrane. In contrast, influenza could still could efficiently enter and infect all non-polarized cells tested after disruption or stabilization of the actin cytoskeleton. To examine the role of the actin motor protein, myosin VI, we expressed a dominant-negative construct in both polarized and non-polarized cells. Influenza virus infectivity in myosin VI tail mutant-transfected cells was significantly decreased in polarized epithelial cells, but not in non-polarized cells. As a whole, our data suggest indispensable roles of a dynamic actin cytoskeleton for influenza virus entry into polarized epithelial cells, a feature not shared with non-polarized cells.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

Redistribution of fibronectin and cytoskeletal proteins during the differentiation of rat mammary tumor cells in vitro

The tumorigenic mammary epithelial stem cell line, Rama 25, is capable of synthesizing and secreting fibronectin but incorporates only small amounts of fibronectin into pericellular material localised in regions of cell-cell and cell-substratum contact. Under certain culture conditions, Rama 25 differentiates into a non-tumorigenic myoepithelial-like cell line, Rama 29, which is capable of retaining fibronectin on the cell surface in characteristic fibrillar formation. The redistribution of fibronectin is accompanied by a reorientation of the cytoskeleton from circular bundles in Rama 25 to parallel arrays of filaments in Rama 29. In vivo, fibronectin is found in the basement membrane of the mammary gland and our in vitro studies lead us to suggest that the mammary myoepithelial cell in vivo synthesizes much of the basement membrane fibronectin.     

Role of serum and hormones during the growth and development of rat mammary tumor epithelial cells in collagen gel culture

Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number and [³H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing 20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [³H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone, prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant stimulatory effect.     

Isolation of rat mammary epithelial cells for polyamine analysis.

Mammary epithelial cells were isolated from either abdominal-inguinal glands or mammary tumours of rats, after enzymic digestion of the tissues, and were analysed for polyamine content. Optimum conditions were developed for the isolation of cells in sufficient yield for the analysis of polyamines from 1 g of mammary gland or 0.5 g of tumour tissue. Complete recoveries of the polyamines in the tissues were achieved in the isolated epithelial cells.     

Transferrin receptor activity in rat mammary epithelial cells

The binding of 125I-transferrin to rat mammary cells isolated by collagenase and hyaluronidase digestion has been investigated. Surface binding was determined at 4 degrees C and total binding also at 4 degrees C but in the presence of 0.1% w/v saponin. KD values between 20 and 25 nM were obtained. Binding assays at 37 degrees C showed the internalisation of the receptor and the bound transferrin was occurring but also provided evidence for an impaired recycling of the receptors to the cell surface in the freshly isolated cells. No differences in total binding were observed in cells prepared at different stages of lactation with a mean value of 29 fmol transferrin bound/micrograms cellular DNA, equivalent to 180,000 receptors per cell.     

Collagen synthesis and deposition during mammary epithelial cell spreading on collagen gels

Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dose-dependent manner. Cytochalasin D inhibition of F-actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.     

Sulphated proteins secreted by rat mammary epithelial cells

The main sulphated proteins secreted by rat mammary gland tissue have Mr of approximately 32 000, 27 000 and 25 000 Da. In addition, there are high Mr components which have a diffuse electrophoretic mobility (Mr > 200 000) and most likely corresponded to proteoglycans. The sulphate groups in the proteins with discrete Mr are most likely all linked to carbohydrates. These sulphated molecules were partially purified and identified to isoforms of rat alpha-lactalbumin for the 25-27 kDa bands and to kappa-casein for the 32 kDa band. This pattern of protein sulphation is, as far as we know, quite specific to rat mammary epithelial cells.     

Role of the cytoskeleton in laminin induced mammary gene expression

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, alpha-casein, and alpha-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of alpha-casein, transferrin, and alpha-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of alpha-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of alpha-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in alpha-casein, alpha-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.     

Oncogene expression in mammary epithelial cells.

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer-promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter-oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of individual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.     

Formation of an actin cytoskeleton and adhesive structures during the spreading of cultured cells

Fibroblast spreading was studied using immunofluorescent method that provided visualization of actin structures and adhesion contacts in the same cell. Four stages of actin system formation were observed. 1. Actin concentration in ruffles at the cell periphery. Formation of numerous dot-like contacts along the whole perimeter of the cell. 2. Formation of a circumferential actin bundle. Focal contacts are located at the outer edge of the bundle. 3. Gradual transformation of the circumferential bundle into actin network with triangular meshes. Peripheral (rather than internal) filaments of the network are associated with the focal contacts. 4. Appearance of the system of long straight actin bundles (stress fibers) associated with dash-like focal contacts. The stress fibers are supposed to arise from the triangular actin network which in its turn arises from the circumferential bundle. It is suggested that the formation of actin cytoskeleton is a process driven by the development of tensions in actin structures attached to the focal contacts at the cell periphery.     

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Reorganization of the apical cytoskeleton of uterine epithelial cells during early pregnancy in the rat: a study with myosin subfragment 1.

Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (S1). MF connections with cytoplasmic organelles and the apical plasma membrane are also described. Transmission electron microscopy revealed that the regular microvilli of non-pregnant, oestrous animals contain several decorated MF with rootlets descending into a densely filamentous terminal web. Following mating, the actin cytoskeleton was examined on days 1, 3 and 6 of pregnancy. In this period, the irregular projections that replace MV assumed an underlying, dense network of decorated MF, whilst smoother surfaces displayed few cytoplasmic filaments. At the time of blastocyst implantation, a structured terminal web was no longer present. Structural details were revealed concerning the contents of large, bleb-like projections found on the apical surface.     

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.