Errata

Scanning electron microscopy of surface morphology of embryonic neural retina cells in suspension revealed striking age-dependent differences between trypsin-dissociated and EGTA-separated cells (EGTA, ethylene bis[oxyethylene nitrolo]tetra-acetic acid). Trypsinized cells were characterized by numerous filopodial processes, ruffles and a relatively smooth-textured cell membrane. EGTA treatment resulted in only partial cell separation, paucity or absence of elongated filopodia, and a coarse-textured cell membrane. The findings are briefly considered in the context of age-dependent differences in retina cell interactions, cell surface properties, and the effects of the two methods of cell separation on the cell surface.     

Scanning electron microscopy of the aggregation of head mesoderm cells from chick embryos

Head mesoderm cells from chick embryos at different stages of development were dissociated and cultured on plastic coverslips. In all cultures several cellular aggregates were described by means of scanning electron microscopy. Isolated cells present filopodia and lamellipodia. However, when mesoderm cells make contact with one another the filopodial and lamellipodial activity in the contact cellular edge disappear. Thus, the cells into cellular clusters do not present projections. The clusters were circular and bidimensional in character. The scanning electron microscopic observations showed that it is the type 1 variant of "contact inhibition of locomotion" which occurs. By means of these mechanisms the bidimensional aggregates are formed and cellular overlapping is not present. Since the behaviour of the mesoderm cells "in vitro" in some way could be comparable to their behaviour "in situ", the results here observed are discussed in relation to the conduct of mesoderm cells "in vivo".     

Specializations in filopodial membranes at points of attachment to the substrate

A mouse cell line (LM), which grows predominantly as spindle-shaped cells with numerous filopodia, was employed in this study. These filopodial projections appear to be important as sites of attachment to the substratum in LM cells. Morphologically the filopodia are slender projections from the cell body which usually attach to the substrate at their distal ends (filopodial footpads). Freeze-fracture of monolayer cultures in situ preserves the spatial relationship of filopodial processes to that of the cell body. Examination of these freeze- fracture preparations reveals a striking difference in the density of intramembrane particles (IMP) in the filopodial-footpad plasmalemma compared with the plasmalemma of the cell body (number of IMP in footpad > cell body). Additionally, there is a marked difference in the number of filipin-sterol complexes on the cell body, compared with the filopodial footpad, implying a difference in the cholesterol content in these regions (filipin-sterol complexes in footpad < cell body). These data suggest a structural and functional specialization of the filopodial-footpad plasma membrane which may be related to cell adhesion.     

Correlation of anionic residue mobility at the filopodial plasma membranes with multilayer formation in transformed 3T3 cells

Simian virus 40-transformed 3T3 (SV40-3T3) cells formed multilayers on a Falcon dish and had numerous filopodial projections, some of which intertwined with those of adjacent cells, in contrast to the few projections of their nontransformed counterparts. When these cells were incubated with polycationic ferritin (0.5 mg/ml), ferritin particles, representative of anionic sites, were spread widely on their surfaces at 4 degrees C, while they formed clusters at 37 degrees C, especially on filopodial surface areas opposing adjacent projections in SV40-3T3 cells. These findings demonstrate an increase in the mobility of molecules with anionic residues on filopodial plasma membranes in SV40-3T3 cells, thus suggesting a role for these projections in the formation of multilayered cell aggregates.     

Tight junctions of dissociated and reaggregated embryonic lung cells

Treatment of embryonic lung tissue with trypsin resulted in clustering of intramembrane particles (IMP) and gradual disassembly of tight junctions. In dissociated single cells kept in trypsin-free medium, IMP are randomly distributed but degradation of tight junctions continue. Vesicles containing tight junction elements were observed within the cytoplasm. It is therefore assumed that tight junctions may be degraded in two ways: breakdown of elements to IMP, and endocytosis. In cells reaggregated by rotation tight junctions reassembled only in hystotypic aggregates. Cycloheximide which interferes with histotypic reaggregation prevents the reassembly of tight junctions.     

A scanning electron microscope study of early sea urchin reaggregation

Sea urchin embryos were observed with SEM during the first 2 h of reaggregation, following dissociation of the 16-cell stage. A dense meshwork, composed of elongated microvilli embedded in the hyaline layer, surrounds the egg during early development. The dissociation procedure strips off some of the meshwork layer leaving fewer and smaller microvilli on the cell surface. Shortly after reaggregation has begun, several types of cell extensions are formed, including filopodia, which anchor the cells to the substrate, and ruffles and pseudopods, which enable the cells to move. Possible factors involved in the behavior of dissociated cells are discussed with regard to (1) the source of additional membrane in the formation of new cell extensions; (2) the ability of the cells to move.     

Effects of puromycin and cycloheximide on properties of cells from Xenopus laevis early embryos

The effects have been studied of puromycin and cycloheximide on the reaggregation of ectoderm cells dissociated from Xenopus laevis blastulae. Puromycin or cycloheximide can inhibit reaggregation, suggesting that cell reassociation is dependent upon protein synthesis. If the cells are allowed a 3 h 'recovery' period in culture medium following dissociation, before being exposed to either puromycin or cycloheximide, higher concentrations of the inhibitors are required to prevent cell aggregation, suggesting that significant synthesis of the proteins required for reaggregation occurs in the 3 h immediately following dissociation. Lower concentrations of puromycin permit cell reaggregation but reduce the normal formation of cilia. The effects have also been observed of puromycin on the scanning electron microscopical appearance of Xenopus blastula ectoderm cells cultured singly in vitro. Puromycin reduces the normal formation of pseudopodia, suggesting that puromycin might inhibit reaggregation partly by inhibiting cell movement. Puromycin also produces some elongated cells, possibly by inhibition of cytokinesis.     

Anti-actomyosin. Influence on adhesive behaviour of eukaryotic cells and of cuvierian tubules

The effect of antisera against chicken gizzard smooth-muscle actomyosin and against pectoralis striated-muscle actomyosin on adhesive behaviour of eukaryotic cells (from sea urchin embryos and from a silicious sponge) and of Cuvierian tubules has been studied. The results with a sea urchin cells, which require divalent cations for aggregation, showed that antiserum to chicken gizzard smooth-muscle actomyosin inhibited reaggregation of trypsin-treated cells better than mechanically dissociated cells, while anti-chicken pectoralis striated-muscle had no effect. Primary reaggregation of trypsin-dissociated sponge cells, in the presence of calcium and magnesium, is also inhibitable by anti-gizzard smooth-muscle but not by anti-pectoralis striated-muscle. Anti-gizzard smooth-muscle had no effect on secondary reaggregation of sponge cells mediated by a soluble aggregation factor. Anti-gizzard smooth-muscle inhibited Cuvierian tubule adhesion.     

Characterization of an extremely motile cellular network in the rotifer Asplanchna spp.

The pseudocoelomic body cavity of the rotifer Asplanchna spp. contains free cells that form a highly dynamic, three-dimensional polygonal network of filopodia. Using video-enhanced differential interference contrast microscopy, we have qualitatively and quantitatively characterized the motion types involved with network motility: (1) filopodial junctions are displaced laterally at 10.52 +/- 0.46 microns/s; (2) free-ending filopodia form and extend at rates of 8.77 +/- 0.40 microns/s, until they retract again at 7.23 +/- 0.87 microns/s; (3) filopodial strands fuse either laterally or tip to the lateral side. The combination of these motion types results in enlargements, diminutions, and extinctions of filopodial polygons, and in the formation of new polygons. Moreover, there is intense and fast (5.11 +/- 0.28 microns/s) particle transport within the filopodial strands. The organization of the cytoskeleton in filopodia was examined by electron microscopy and by labeling with fluorescent-tagged phalloidin. Filopodia contain several microtubules that are often organized in a bundle. Moreover, F-actin is present within the filopodia. To characterize which of these cytoskeletal systems is involved with cell and organelle motility, we have examined cell dynamics after incubations with colchicine or cytochalasin D. The results of these pharmacological experiments provide evidence that microtubules are required for both cell and organelle motility, but that actin filaments contribute to these phenomena and are required for the structural maintenance of slender filopodia.     

Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.     

Some effects of trypsin dissociation on the inhibition of reaggregation among embryonic chicken neural retina cells by cycloheximide

The effects of cycloheximide on the reaggregation of trypsin-dispersed, embryonic chicken, neural retina cells were investigated. The cells were used either immediately after isolation (F-cells), or after 24 hr prior culture under conditions not permitting reaggregation (V-cells). The parameters of aggregation used were the size of aggregates formed in gyrotatory shaker cultures, and the concentration of single cells in these cultures. At both 24 and 48 hr, following treatment with cycloheximide, the mean diameters of the aggregates of F-cells showed a greater reduction than the V-cells, when compared with their corresponding controls. In addition, cycloheximide resulted in a higher concentration of single cells, that is less cell/cell adhesion, than in comparable controls. A higher proportion of single cells were present in the F-cell cultures in the presence of cycloheximide than in the V-cell cultures. Thus, the degree of inhibition of cell adhesion by one inhibitor of protein synthesis differed in F-cells and V-cells. These experiments may serve to focus attention on some secondary effects of tryptic dissociation, which is an often overlooked factor in subsequent studies of reaggregating embryonic cells, particularly in those experiments involving the use of metabolic inhibitors.     

Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.     

A comparative study of collagenase- and trypsin-dissociated embryonic heart cells: reaggregation, electrophysiology, and pharmacology

The electrophysiological and pharmacological properties of aggregates prepared from cells of 7-day-old chick embryo heart ventricles depend on the enzyme used for cell dissociation. The mean beat rate of aggregates formed from trypsin-dissociated cells was about 53 beats/min whereas aggregates formed from collagenase-dissociated cells had a mean beat rate of more than twice this value. Spontaneous activity of most aggregates formed from trypsin-dissociated cells was inhibited by elevating external potassium or by adding tetrodotoxin to the medium. A similar response to potassium was seen in all aggregates formed from collagenase-dissociated cells. However, approximately half of the aggregates formed from collagenase-dissociated cells were tetrodotoxin insensitive. Intracellular microelectrode recordings demonstrated that aggregates formed from collagenase-dissociated cells typically had reduced action potential maximal upstroke velocities and depolarized threshold potentials in comparison to those recorded from aggregates formed from trypsin-dissociated cells. In the presence of tetrodotoxin the maximal upstroke velocity of aggregates formed from either collagenase- or trypsin-dissociated cells decreased markedly. In the case of the collagenase-treated cells, the spontaneous activity which persisted in the presence of tetrodotoxin was abolished by the slow channel blocker D-600. Computer simulation of membrane depolarization supports the view that aggregates formed from collagenase-treated cells have a reduced fast inward sodium current and a significant leakage current. Aggregates prepared from trypsin-dissociated cells display properties which more closely resemble those of intact 7-day embryonic ventricular tissue. We therefore conclude that, contrary to previous reports, collagenase is not the enzyme necessarily best suited for cell dissociation in all tissue culture studies.     

Sponge cell reaggregation: Mechanisms and dynamics of the process

Sponges (Porifera) are lower metazoans whose organization is characterized by a high plasticity of anatomical and cellular structures. One of the manifestations of this plasticity is the ability of sponge cells to reaggregate after dissociation of tissues. This review brings together the available data on the reaggregation of sponge cells that have been obtained to date since the beginning of the 20th century. It considers the behavior of dissociated cells in suspension, the mechanisms and factors involved in reaggregation, and the rate and stages of this process in different representatives of this phylum. In addition, this review provides information about the histological structure of multicellular aggregates formed during reaggregation of cells and the regenerative morphogenetic processes leading to the formation of normal sponges from these multicellular aggregates.     

Steric exclusion of cells. A mechanism of glycosaminoglycan-induced cell aggregation.

The mechanism by which chondroitin sulfate enhances both the self-aggregation and the concanavalin A (ConA)-induced agglutination of trypsin-dissociated embryonic chick retina cells was investigated. Studies with fluorescently labeled ConA showed no influence of chondroitin sulfate on patching or capping. When ³H- or ³⁵SO₄-labeled glycosaminoglycans or proteoglycans from retinas were added to freshly dissociated cells, an average of less than 2% of the label became associated with the unwashed cell pellet, and most of this was removed from the cells by a single wash. The presence of ConA did not alter the amount of binding by this criterion. Rapid cell aggregation in the absence of ConA was promoted by a number of natural and synthetic polymers. Aggregation rate bore a direct relationship to polymer viscosity at low viscosities and was inhibited at high viscosities, apparently due to reduced cell collision frequency. For any given polymer, aggregation was directly related to its molecular weight and concentration. Linear polymers were more effective than branched ones. Neutral polymers were as effective as those which were strongly polyanionic. Stable aggregates of formalin-fixed cells were not promoted by polymers. All of these observations are consistent with the hypothesis that enhancement of retina cell aggregation by physiological concentrations of glycosaminoglycans is due largely to steric exclusion of the cells by the polymer mesh. Although others have shown that glycosaminoglycans probably interact specifically with some cells, the evidence presented here suggests that these macromolecules by virtue of their excluded volumes could also have important non-specific influences on cell migration and cell reorganization during morphogenesis.     

Filopodia are induced by aquaporin-9 expression

Understanding filopodial formation in motile cells is a pertinent task in cell biology. In the present study we show that expression of the human water channel aquaporin-9 (AQP9) in different cell lines induces the formation of numerous filopodial extensions. Several lines of evidence support the role of aquaporins functioning both as a water channel and signaling participant. The number of filopodia is decreased by site-directed serine substitutions in putative PKC-binding or -phosphorylation sites at amino acid position 11 and 222 in AQP9. The filopodial phenotype obtained with wild-type AQP9 is associated with elevated levels of active Cdc42, while serine-deleted mutants have reduced levels of GTP-Cdc42. Co-transfection with inhibitory N-WASP CRIB completely abolishes wild-type AQP9-induced filopodia formation. Active PKC(zeta) phosphorylates wild-type AQP9 and myristoylated PKC(zeta) pseudosubstrate inhibits the formation of filopodia in AQP9-expressing cells. Expression of wild-type AQP9, but not mock or serine substituted mutants, increases sensitivity to hypo-osmolaric conditions, yielding a rapid morphological rounding of cells and cell death starting as early as 24 h post-transfection. We propose that increased water influx through AQP9 is critically involved in the formation of membrane protrusions, and that AQP9-induced actin polymerization is augmented by activation of Cdc42 and PKC(zeta).     

Freeze-fracturing and surface labelling of embryonic neural retina cells

Freeze-fracturing of dissociated and aggregating neural retina cells from 7-day chick embryos revealed on the inner faces (PF) of the cell membrane numerous particles 6–20 nm in size. In contrast, the PF faces of blebs and some of the lobopodia that project from the cell surface were practically devoid of such particles. However, the elongated filopodia that abound on these cells showed numerous particles on their PF faces. These regional differences in the distribution of particles on PF faces of these cells are interpreted as reflecting membrane activity that leads to the formation of blebs and lobopodia. The frequent presence of “pits” at the basis of blebs and lobopodia is described. It is suggested that the “pits” are associated with the formation of these membrane projections; they may represent anchoring sites for microfilaments and for microtubules involved in the dynamic structure of the cell surface. ConA-binding sites on these cells were studied by scanning electron microscopy, using labeling with hemocyanin. The distribution of these sites on different regions of the cell surface coincided with the regional differences in the distribution of the inner membrane particles.     

Visualization of cellular aggregates cultured on a three dimensional collagen sponge matrix

Summary A one-step vital stain is described for the macroscopic visualization of histotypic cell aggregates in fetal rat lung organotypic cultures. Organotypic cultures are incubated in 0.05-0.1% 2,3,5′-triphenyl tetrazolium chloride (TTC) in culture medium (37°C). Living cells reduce the tetrazole to a water-insoluble red colored formazan. Cell aggregates appear as densely stained foci against the lighter background of the Gelfoam substrate. Stained cultures may be scanned macroscopically to determine the degree of reaggregation and assess cell viability. Identification of aggregates by TTC staining improves the efficiency of tissue processing for electron microscopy and does not alter the ultrastructural appearance of the cultured cells. This work was funded in part by the United Cerebral Palsy Research and Educational Foundation, Inc. and the National Heart, Lung, and Blood Institute (Grants 1ROHL19513 and 1 R01HL21008).     

Transfection of Reaggregating Embryonic Chicken Retinal Cells with an Antisense 5'-DNA Butyrylcholinesterase Expression Vector Inhibits Proliferation and Alters Morphogenesis

Abstract: The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.     

Enzymatic dissection of embryonic cell adhesive mechanisms

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.