Spreading of mouse fibroblasts on the substrate with multiple spikes

Mouse embryo fibroblasts were cultivated on special substrates with discontinuous surfaces. The substrates were silicon plates with multiple vertical (65-90 microns height) spike-like silicon microcrystals evenly distributed on the plate surfaces. It was shown that the cells were successfully spread and flattened on these substrates. The spread cells formed several discrete attachment zones at the tops and side surfaces of the spikes; these zones were separated from one another by distances considerably greater than the diameter of the unspread cell. At early stages of spreading the unspread cells attached to the tops of single spikes and extended long filopodia attached to the distant spikes. At later stages the lamellae were formed between the filopodia: probably these filopodia served as guidelines for extension of lamellae and progressive cell spreading. These experiments demonstrated that continuity of substrate surface is not a necessary condition for advanced cell spreading.     

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.     

Comparative study, using fluorescent methods, of cell membranes and their reconstituted liposomes

Cell plasma membranes and proteoliposomes reconstituted from solubilized plasma membranes of thymocytes and Ehrlich ascites carcinoma cells have been studied by fluorescent methods. It has been shown that proteoliposomes are characterized by greater polarization and rigidity of microsurroundings in membrane proteins and greater microviscosity of membrane lipids. Proteoliposomes from thymocyte membranes contain less membrane proteins and express lower polarity of the lipid bilayer than proteoliposomes from Ehrlich ascites cells.     

Adhesion defect of ascites cells corrected with membrane-bound attachment molecules

Adhesion is obligatory for cell proliferation in most types of cells. This function becomes defective after malignant transformation. An extreme example is ascites cells which proliferate in suspension. The nature of their defects remains obscure. Here we show that the linking of biotin molecules to the Ehrlich ascites carcinoma cells enables these cells to spread normally on an avidin-coated substrate. The spreading was the result of specific avidin-biotin interaction. The morphology of the spread cells and sensitivity to different inhibitors are similar to those of normal epithelia. Thus it is enough to supply appropriate substrate-adhesive molecules to the ascites cell surface to normalize their adhesion.     

Lectin-induced cell agglutination and membrane cholesterol levels

The membranes of human and guinea pig erythrocytes were enriched with, or depleted of cholesterol. Ehrlich ascites carcinoma cells were also enriched with cholesterol and the extra slerol shown to be present in the plasma membrane. Enrichment of the cells with sterol made them less susceptible to agglutination by concanavalin A (ConA) or phytohemagglutinin (PHA), while removal of sterol from the erythrocytes increased their susceptibilily to agglutination. It is suggested that following changes in surface membrane sterol levels there are changes both in short-range movement of individual receptor molecules and in cell shape and deformability which control the agglutinability of the cells.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Interleukin-1 is a motility factor for human breast carcinoma cells in vitro: additive effect with interleukin-6.

Interleukin-1 beta (Il-1 beta) and interleukin-1 alpha (Il-1 alpha) were shown to act as motility factors for the human breast carcinoma cell lines SK-BR-3 and ZR-75-1 in vitro. Both cytokines induced transition from the stationary to the motile phenotype (spreading). Il-1 beta stimulated translocation, shape change and random migration (chemokinesis) of SK-BR-3 cells as demonstrated by time-lapse video recordings and by a modified Boyden chamber assay. Interleukin-6 (Il-6) stimulated spreading of the SK-BR-3 cells; an additive effect with Il-1 beta on spreading and fast plasma membrane movements was evidenced. In the SK-BR-3 cell line, the signal transduction of Il-1 beta and Il-6 differed, since only the effect of Il-6 on spreading was sensitive to pertussis toxin. Both Il-1 beta and Il-6 required protein synthesis to stimulate spreading, since cycloheximide inhibited the effect of the cytokines. Induction of an autocrine loop of Il-6 in the SK-BR-3 cells by Il-1 beta was unlikely, since after stimulation with Il-1 beta, no induction of Il-6 activity was measured, nor was inhibition of stimulated spreading seen in the presence of an antiserum against Il-6. Addition of Il-8 or of an antiserum against Il-8 did not affect spreading. We concluded that Il-1 and Il-6 could act as motility factors for human breast carcinoma cells, in both an independent and an additive way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Constitutive endocytosis and recycling of the neuronal glutamate transporter, excitatory amino acid carrier 1

The neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has a diverse array of physiologic and metabolic functions. There is evidence that there is a relatively large intracellular pool of EAAC1 both in vivo and in vitro, that EAAC1 cycles on and off the plasma membrane, and that EAAC1 cell surface expression can be rapidly regulated by intracellular signals. Despite the possible relevance of EAAC1 trafficking to both physiologic and pathologic processes, the cellular machinery involved has not been defined. In the present study, we found that agents that disrupt clathrin-dependent endocytosis or plasma membrane cholesterol increased steady-state levels of biotinylated EAAC1 in C6 glioma cells and primary neuronal cultures. Acute depletion of cholesterol increased the V(max) for EAAC1-mediated activity and had no effect on Na(+)-dependent glycine transport in the same system. These agents also impaired endocytosis as measured using a reversible biotinylating reagent. Co-expression with dominant-negative variants of dynamin or the clathrin adaptor, epidermal growth factor receptor pathway substrate clone 15, increased the steady-state levels of biotinylated myc-EAAC1. EAAC1 immunoreactivity was found in a subcellular fraction enriched in early endosome antigen 1 (EEA1) isolated by differential centrifugation and partially co-localized with EEA1. Co-expression of a dominant-negative variant of Rab11 (Rab11 S25N) reduced steady-state levels of biotinylated myc-EAAC1 and slowed constitutive delivery of myc-EAAC1 to the plasma membrane. Together, these observations suggest that EAAC1 is constitutively internalized via a clathrin- and dynamin-dependent pathway into early endosomes and that EAAC1 is trafficked back to the cell surface via the endocytic recycling compartment in a Rab11-dependent mechanism. As one defines the machinery required for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression.     

A Unified Kinetic Hypothesis of Carrier-Mediated Transport: Its Applications

A model analysis of the process of carrier mediated membrane transport is presented, wherein the carrier is present in two forms of differing affinity for substrate. The two forms of carrier undergo interconversion by asymmetric metabolic reactions on each side of the membrane. From this model system expressions are derived for the steady-state distribution ratio for substrate, for the unidirectional fluxes of substrate and hence for the initial velocity of uptake of substrate, and for the effect of preloading cells upon the initial velocity of uptake of labeled substrate. These expressions are applied to published data for glycine transport in Ehrlich ascites tumor cells to obtain numerical values for the parameters of a concentrative membrane carrier system. Concentrative uptake is shown to be consequent to the differing affinities of the two forms of carrier. When the affinities of the two forms are equal, equilibrative uptake occurs. The model analysis is applied to the phenomena of metabolic and competitive inhibition.     

The nonspecific metabolic reaction of cells to extreme exposures

This work summarizes the authors’ recent investigations into metabolic changes in blood lymphocytes, cancer cells, and hepatocytes during tumor growth from mice with Ehrlich ascites carcinoma. These results were compared with the metabolic changes in hepatocytes from rats after hyperthermia and in lymphocytes from patients with different diseases. It was shown that the extreme conditions induced metabolic changes that were independent of the cell type or the nature of the extreme factor. These changes characterized the metabolic mechanisms of cell adaptation and disadaptation. The concept of the “nonspecific metabolic reaction” of cells to extreme exposures had been introduced.     

The effects of cell-cell contact on the spreading of pigmented retina epithelial cells in culture

The behaviour of chick embryo pigmented retina epithelial (PRE) cells has been studied in living and fixed cultures. Isolated PRE cells lacking contacts with other cells were characteristically only poorly spread upon the substrate, blebbed vigorously and lacked leading lamellae. PRE cells incorporated into islands or sheets of cells were extensively spread upon the substrate, lacked blebs and displayed typical leading lamellae if marginally positioned in an island. Observations of living cultures demonstrated that within 3 h of establishing contact with an island of cells a previously isolated PRE cell lost the morphology characteristic of isolated cells and became indistinguishable from its neighbours in the island. Measurements of the area of substrate occupied by single cells and cells in 2-cell islands suggests that similar changes occur as two cells make contact to form a 2-cell island. The evidence suggests that these changes are a direct response to the establishment of a cell-cell contact and I propose that the phenomenon be termed ‘contact-induced spreading’.Contact-induced spreading is not an ‘all or none’ phenomenon since isolated PRE cells can spread extensively and cease blebbing in the absence of cell contact. However a given isolated PRE cell spends only a very small proportion of its time displaying this well spread morphology and therefore at any time the majority of isolated PRE cells display the poorly spread morphology.The possible relationship between contact-induced spreading and other cellular interactions known to be dependent on cell-cell contact is discussed.     

Alterations in biosynthesis and homeostasis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [14C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [14C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

Alterations in biosynthesis and homeostatis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [¹⁴C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [¹⁴C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.     

Blebbing dynamics during endothelial cell spreading

Cell spreading is a critical component of numerous physiological phenomena including cancer metastasis, embryonic development, and mitosis. We have previously illustrated that cellular blebs appear after abrupt cell-substrate detachment and play a critical role in regulating membrane tension; however, the dynamics of bleb-substrate interactions during spreading remains unclear. Here we explore the role of blebs during endothelial cell spreading using chemical and osmotic modifications to either induce or inhibit bleb formation. We track cell-substrate dynamics as well as individual blebs using surface sensitive microscopic techniques. Blebbing cells (both control and chemically induced) exhibit increased lag times prior to fast growth. Interestingly, lamellae appear later for blebbing compared to non-blebbing cells, and in all cases, lamellae signal the start of fast spreading. Our results indicate that cellular blebs play a key role in the early stage of cell spreading, first by controling the initial cell adhesion and then by presenting a dynamic inhibition of cell spreading until a lamella appears and fast spreading ensues.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 × 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Studies on cell surface conformation following injury. II. Scanning and transmission electron microscopy of cell surface changes following anoxic injury in Ehrlich ascites tumor cells.

Exposure of Ehrlich ascites tumor cells to anoxia resulted in rapid and characteristic conformational changes of cell surface topography. Combined scanning and transmission E/M studies revealed rapid alterations including simplification of the cell surface configuration with disappearance of microvilli which were replaced with formation of blebs and recesses at the cell periphery. These surface changes were accompanied by characteristic organelle alterations inside the cells which in this and other cellular systems have been shown to be reversible. Later, the cell surface topography became smoother and monotonic with small blebs and cribriform invaginations in addition to larger eruptions of the cell periphery. Combined transmission E/M studies revealed fragmentation of cellular membrane systems and lysis of organelles indicating the irreversible phase of anoxic injury. The rapid conformational surface changes encountered in Ehrlich ascites tumor cells following anoxia suggest the important role of the plasma membrane and its unfolding as a virtually instantaneous response of the cells to this injury.     

Freeze-fracture study of plasma membranes in wild type and daunorubicin-resistant Ehrlich ascites tumor and P388 leukemia cells

The plasma membrane is considered to play a major role in the development and maintenance of the multidrug resistance (MDR) phenotype, a role which may in part be mediated by an inducible 170 kD transmembrane protein (P-170). The present freeze-fracture study of plasma membranes of daunorubicin-resistant Ehrlich ascites and P388 leukemia cells demonstrated a significant increase in the density of intramembrane particles (IMP) in the P-face, but not the E-face, of resistant sublines compared with wild type cells. Furthermore, a three-dimensional histogram plot of the diameters of P-face IMPs in Ehrlich ascites tumor cells showed the emergence of a subpopulation of 9 X 11 nm IMPs not found in wild type cells. The size of these IMPs would be consistent with a MW of approximately 340 kD, thus indicating that P-170, shown to be present in both resistant cell lines by Western blot analysis and immunohistochemical staining, exists as a dimer in the plasma membrane. Incubation with the calcium channel blocker verapamil, in concentrations known to inhibit daunorubicin efflux in resistant cells, showed evidence of membrane disturbance in the form of IMP clustering in both wild type and resistant Ehrlich ascites tumor cells. However, incubation with daunorubicin itself did not alter the freeze-fracture morphology of the plasma membranes.     

Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.