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A scanning electron microscope study of cell shape and cell appendages in the primitive streak region of the rat and chick embryo.

This report provides a scanning electron microscopic account of cell shapes and cell appendages that are observed in the primitive streak region of the rat and chick embryos. Epiblast cells become progressively elongated in the primitive streak region until flask cells predominate medially. The flask cells have a broad basal end directed toward the endoderm. In addition to fine filopodia, broad lamellipodia are found anchoring the flask cells to subjacent cells. The primary mesenchyme cells are at first round in shape and closely packed, but laterally are flattened and more dispersed. The mesenchyme cells are associated with each other by filopodia and lamellipodia and with the epiblast and endoderm by filopodia. On the basis of this description it is suggested that cell movement through the primitive streak occurs by cell extension, attachment by basal lamellipodia, and cell shortening that results in the movement of individual cells in a cell stream.

Animals

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions.

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.

Animals

Detection of X-ray damage repair by the immediate versus delayed plating technique is dependent on cell shape and cell concentration.

A method commonly used to measure the ability of cells to repair potentially lethal damage (PLD) is to compare immediate plating (IP) and delayed plating (DP) survival. Lower cell survival under IP conditions relative to that after DP conditions has been interpreted to indicate a higher ability of cells to repair potentially lethal damage (PLD) under DP conditions. However, this IP radiosensitization has not been observed in several cell lines and tumor models. IP conditions involve treatment of cells with trypsin and plating them into fresh growth medium. We have investigated the possibility that radiosensitization under IP conditions may be related to both the cell-shape and the nutrient concentration in growth medium (GM, MEM + 15% serum). This idea predicts that the IP and DP survival of spheroids will show a response similar to the IP survival of cells in monolayers and that the IP and DP survival of crowded monolayer cells in high densities will be the same. Chinese hamster V79 cells grown in monolayers (spread cells) and spheroids (clumps of round cells) were used. The IP survival was lower than the DP survival for spread log phase monolayer cells but not for round log phase cells in spheroids. Radiosensitization of cells by fresh (as opposed to spent) growth medium was absent for high density plateau phase cells in monolayers at or above 2 x 10(6) cells/ml. However, PLD repair could be demonstrated in spheroid cells and in high density plateau phase cultures by exposing cells to hyperthermia or hypertonic saline. Comparison of immediate plating versus delayed plating survival detects PLD repair only in well spread low density monolayer cells, but not in round spheroid cells nor in dense monolayer cells at > 10(7) cells/25 cm2 flask/5 ml medium. The absence of a difference between IP and DP cell survival does not mean that PLD repair is absent. Incorrect prediction of tumor response to radiotherapy can occur when PLD repair capacity is assayed as a ratio of DP/IP survival. More than one method must be used to measure the capacity of cells to repair their PLD.

Animals

Protein kinase C activity in erythrocytes in primary hypertension: regulation of cell shape and cation transport.

Protein kinase C activity in the lysate of erythrocytes of patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR) was found to be increased by 1.6-2.0 times as compared with normotensive controls. Membrane cytoskeleton alterations observed in the erythrocytes of patients with EH and SHR were revealed in decreased average erythrocyte volume, increase of cup-shaped cell formation, and increase of basal phosphorylation of band 4.9 protein. In addition, the rate of Na(+)-H+ exchange in erythrocytes of EH patients and SHR was increased by 1.9-fold. In vitro treatment of erythrocytes of healthy donors and Wistar-Kyoto rats (WKY) with protein kinase C activator (12-O-tetradecanoylphorbol-13-acetate) leads to similar changes of cell shape, cell volume, band 4.9 protein phosphorylation and Na(+)-H+ exchange, as well as to an increase of diS-C3-(5) fluorescence. It may be assumed that alterations of these parameters revealed in primary hypertension are caused by increased activity of protein kinase C.

Adult

Morphology of an Escherichia coli mutant with a temperature-dependent round cell shape.

Mutants of Escherichia coli capable of growing in the presence of 10 microgram of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 30 degrees C but grew as spherical cells at 42 degrees C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip+ rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 30 degrees C but relatively resistant to mecillinam at 42 degrees C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 30 degrees C in reduced amounts and was undetectable in the membranes of cells grown at 42 degrees C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 42 degrees C, although it grows with normal rodlike morphology at 30 degrees C. At 42 degrees C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.

Amdinocillin

The human progenitor cell antigen (CD34) is localized on endothelial cells, dermal dendritic cells, and perifollicular cells in formalin-fixed normal skin, and on proliferating endothelial cells and stromal spindle-shaped cells in Kaposi's sarcoma.

The human progenitor cell antigen (CD34) is selectively expressed on hematopoietic progenitor cells in the bone marrow. In either cryostat sections of snap-frozen skin, or formalin-fixed paraffin-embedded sections of normal skin, anti-CD34 monoclonal antibody immunostained vascular endothelial cells and perivascular/interstitial dendritic cells, particularly in the reticular dermis. A distinctive population of perifollicular spindle-shaped cells in the midportion of the follicle (ie, bulge area), which is the site of the putative hair follicle stem cells, were CD34 positive, as were spindle-shaped cells in and around the eccrine glands accentuating their basement membrane zone. In patch/plaque--and tumor-stage acquired immunodeficiency syndrome-associated Kaposi's sarcoma lesions, CD34 expression was present on both the proliferating endothelial cells as well as the spindle-shaped stromal cells. CD34 positive endothelial cells and spindle-shaped stromal cells may play important participatory and supportive functions in both normal and diseased skin.

Antigens, CD

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape.

The major cell surface glycoprotein of chick embryo fibroblasts, cellular fibronectin (formerly known as CSP or LETS protein), was purified and used to produce monospecific antisera. After affinity purification, the anti-fibronectin was used to investigate fibronectin's localization, its transfer from intracellular to extracellular pools, its antibody-induced redistribution on the cell surface, and its role in cell shape. Anti-fibronectin localizes to extracellular fibrils located under and between sparse cells, and to a dense matrix that surrounds confluent cells. Cellular fibronectin is also present in granular intracytoplasmic structures containing newly synthesized fibronectin before secretion. This intracellular staining disappears 2 h after treatment with cycloheximide or puromycin, and returns after removal of these protein synthesis inhibitors. In pulse-chase experiments using cycloheximide, fibronectin was sequentially transferred from the intracellular to the fibrillar extracellular forms. Transformation of chick fibroblasts results in decreases in both extracellular and intracellular fibronectin, and in altered cell shape. Treatment of untransformed chick fibroblasts with anti-fibronectin results in rapid (30 min) alteration to a rounder cell shape resembling that of many transformed cells. These rapid shape changes are followed by a slow, antibody-induced redistribution of fibronectin to supranuclear caplike structures. This "capping" is inhibited by metabolic inhibitors. Reconstitution of cell surface fibronectin onto transformed cells restores a more normal fibroblastic phenotype. The reconstituted fibronectin on these cells organizes into fibrillar patterns similar to those of untransformed cells. As with untransformed cells, treatment of these reconstituted cells with anti-fibronectin also results in cell rounding and "capping" of fibronectin.

Animals

Regulation of C-myc and C-Ha-ras oncogene expression by cell shape.

The influence of cell shape on the expression of proto-oncogenes was examined in normal and malignant human cells that varied in their sensitivities to contact-inhibition of proliferation. Cells were constrained into varying degrees of roundness by plating onto culture surfaces coated with different concentrations of poly(2-hydroxyethyl methacrylate) (poly[HEMA]) and assayed for proliferation capacity and levels of c-myc, c-ras, c-fos, and c-fes mRNAs. Proliferation of contact-inhibited normal CUA-1 fibroblasts and the variant HT-IFNr cells was highly coupled to cell shape. As these cells became more rounded, a critical degree of roundness was reached at which proliferation ceased. In contrast, proliferation of non-contact-inhibited malignant HT-1080 cells was independent of cell shape. Northern analysis revealed that expression of c-myc and c-ras was highly sensitive to cell shape in the normal CUA-1 cells but not in the malignant HT-1080 or variant HT-IFNr cells. Levels of c-myc and c-ras mRNAs declined to nearly undetectable levels in CUA-1 cells at degrees of roundness that correlated with loss of proliferative ability. Expression of c-fos and c-fes oncogenes were independent of cell shape in all cells tested. Quantification of transcription rates by the nuclear run-off assay showed that shape modulation of c-myc and c-ras oncogene expression occurred at the transcriptional level. These data suggest that changes in cell shape can modulate expression of certain oncogenes and that these changes correlate with the cell's ability to proliferate. Moreover, inability to regulate c-myc and c-ras oncogene expression is associated with loss of shape-dependent growth controls and contact inhibition but that loss of this regulation alone is not sufficient to release cells from contact-inhibited controls.

Cell Division

The human erythrocyte membrane skeleton may be an ionic gel. II. Numerical analyses of cell shapes and shape transformations.

In the first paper in this series (Stokke et al. Eur Biophys J 1986, 13:203-218) we developed the general theory of the mechanochemical properties and the elastic free energy of the protein gel--lipid bilayer membrane model. Here we report on an extensive numerical analysis of the human erythrocyte shapes and shape transformations predicted by this new cell membrane model. We have calculated the total elastic free energy of deformation of four different cell shape classes: disc-shaped cells, cup-shaped cells, crenated cells, and cells with membrane invaginations. We find that which of these shape classes is favoured depends strongly on the spectrin gel osmotic tension, IIGu, and the surface tensions, IIEu and IIPu, of the extracellular and protoplasmic halves of the membrane lipid bilayer, respectively. For constant ratio IIEu/IIPu greater than O large negative or positive values of IIGu favour respectively the crenated and invaginated cell shape classes. For small absolute values of IIGu, IIEu, and IIPu, biconcave or cup-shaped cells are the stable ones. Our numerical analysis shows that the higher the membrane skeleton compressibility is, the smaller are the values of IIGu needed to induce cell shape transformation. We find that the stable and metastable shapes of discocytes and stomatocytes generally depend both on the shape of the stressfree membrane skeleton and the membrane skeleton compressibility.

Elasticity

Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids.

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.

Animals

A cytoskeletal spring for the control of cell shape in outer hair cells isolated from the guinea pig cochlea.

A two-dimensional cortical cytoskeletal lattice associated with the lateral plasma membranes of mammalian outer hair cells maintains cell shape and provides a restoring force to oppose active changes in cell length. The lattice is composed of two morphologically distinct filaments which are arranged to reinforce the cell circumferentially whilst allowing limited changes in cell length and diameter. This function can only be fulfilled if intracellular pressure is high enough to put the lattice under tension.

Animals

Development of spindle-shaped cells and chondroid cells from androgen-dependent Shionogi carcinoma 115. A light and electron microscopic study.

Androgen-dependent Shionogi carcinoma 115 (SC115) is an undifferentiated medullary carcinoma consisting of compact round cells. However, when host male DS mice were castrated 2 weeks after tumor transplantation, tumors composed of compact round cells, spindle-shaped cells and chondroid cells grew 4 weeks after castration. Compact round cells with desmosomes were arranged in solid nests and exhibited immunoreactivity for keratin protein. Spindle-shaped cells had prominent rough endoplasmic reticulum, and appeared to secrete collagen. Chondroid cells had the characteristics of chondrocytes. The light and electron microscopic features were highly suggestive of a transition from compact round cells to spindle-shaped cells, and from spindle-shaped cells to chondroid cells. The histology of this tumor thus suggests that SC115 cells are able to change into chondroid cells via spindle-shaped cells.

Androgens

Changes in cell shapes and cytokeratins of epithelial cells during the infiltration of lymphocytes in the human palatine tonsils.

By the immunohistochemical method using anti-keratin antibodies (KL1-antigen, 56 KD keratin; PKK1-antigen, 40-52.5 KD keratin) and electron microscopy, the changes in cell shapes and the cytokeratin components of the epithelial cells during the infiltration of lymphocytes were studied. In the surface epithelium, PKK1 reacts with only the keratinocytes in the basal layer, while KL1 stains in the spinous layer. In the neck portion of the crypt, transformed keratinocytes scattered in the spinous layer react intensely with PKK1. These cells issue prolonged cytoplasmic processes which surround the cavities filled with infiltrating lymphocytes. In the deep portion of the crypt, PKK1-positive cells interconnect with one another constituting the network of the star-shaped reticular cells. The enlarged intercellular spaces of the reticular cells are filled with many lymphocytes. Our observations suggest that the infiltration of lymphocytes into the crypt epithelium induces the change in keratin expression of the epithelial cells in the human palatine tonsils, and the change of keratin molecules makes the stratified flattened epithelium to transform into the network of star-shaped reticular cells.

Child

Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Transitional Cell

Glutaraldehyde induces cell shape changes in isolated outer hair cells from the inner ear.

Individual isolated outer hair cells (OHCs) from the cochlea were maintained in a collagen gel and viewed in the light microscope. They were observed during fixation and processing for transmission electron microscopy and individual cells were selected for observation in the electron microscope. Application of glutaraldehyde at several concentrations caused OHCs to become shorter. Shrinkage occurred during dehydration but there was no further change during infiltration with the epoxy resin. Ultrastructural analysis of isolated cells fixed with glutaraldehyde and postfixed with osmium tetroxide showed that these cells were similar to cells fixed in the intact cochlea. The glutaraldehyde-induced cell shape change is similar to the shortening seen in intact OHCs in response to the application of solutions containing high potassium or caffeine. Application of glutaraldehyde to cells pretreated with potassium or caffeine caused further shortening. Glutaraldehyde-induced cell shape change was not blocked by the application of tetracaine, which did prevent potassium-induced and caffeine-induced shortening. Glutaraldehyde-induced cell shape change was not stopped by short treatment with N-ethylmaleimide, which did inhibit potassium-induced shortening. Results from these experiments suggest that the glutaraldehyde-induced OHC shape change is not caused by an effect on the membrane or by calcium activation of a contractile response. Shortening may be caused by shrinkage due to cross-linking of proteins.

Aldehydes

Effect of cell shape on proteinase secretion by epithelial cells.

Cell proliferation has been found to correlate with increased secretion of proteinases, such as plasminogen activator, in several different cell populations. In addition, the shape of the cell may also play a role in regulating proteinase secretion. However, the relationship between cell proliferation, cell shape and proteinase secretion has not been studied in diploid epithelial (E) cells cultured from porcine periodontal ligament (PL). We have modified PLE cell shape by physical means, such as growth on less-adhesive substrata and mechanical stretching, and by exposure to cholera toxin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Neutral proteinase and plasminogen activator secretion were found to correlate with cell shape, the more round the cells, the greater the amount of proteinase secreted. PLE cells, stimulated to proliferate by cholera toxin or dibutyryl cyclic AMP, were more spread than control cells, but secreted less neutral proteinase and plasminogen activator. TPA stimulated cell proliferation slightly but, in contrast to cholera toxin, increased cell rounding and the secretion of neutral proteinase and plasminogen activator. Thus proteinase secretion was related more to cell shape than to cell proliferation.

Animals