Possible roles for cell-to-substratum adhesion in neuronal morphogenesis

The role of cell-to-substratum adhesion in the initiation, elongation, and branching of axons from embryonic sensory neurons was investigated. Cells from sensory ganglia of 4–8-day-old chicken embryos were cultured on several substrata: including collagen; polyornithine-, polylysine-, and polyglutamate-coated surfaces, and tissue culture dishes. The air-blaster method was used to measure growth cone-substratum adhesion.Growth cones adhere much more strongly to polyornithine- or polylysine-coated surfaces and to the upper surfaces of glial cells than to tissue culture plastic. Axons, too, adhere tightly to these substrata, and are crooked, whereas on tissue culture plastic, axons are not adherent and are straight. The fraction of neurons that form axons and the rates of axonal elongation and branching are markedly increased when cells are cultured on polyornithine-coated dishes as compared to tissue culture dishes.This correlation of strong adhesion and enhanced neuronal morphogenesis suggests that adhesive interactions between the growth cone and the microenvironment in an embryo are crucial parts of the initiation and elongation of neuronal processes. Regulation of neuronal morphogenesis may be expressed through the physicochemical properties of the interacting cell surfaces and extracellular environment.     

Bubble Contact Angle Method for Evaluating Substratum Interfacial Characteristics and Its Relevance to Bacterial Attachment

A bubble contact angle method was used to determine interfacial free-energy characteristics of polystyrene substrata in the presence and absence of potential surface-conditioning proteins (bovine glycoprotein, bovine serum albumin, fatty acid-free bovine serum albumin), a bacterial culture supernatant, and a bacterial exopolymer. Clean petri dish substrata gave a contact angle of 90°, but tissue culture dish substrata were more hydrophilic, giving an angle of 29° or less. Bubble contact angles at the surfaces exposed to the macromolecular solutions varied with the composition and concentration of the solution. Modification by pronase enzymes of the conditioning effect of proteins depended on the nature of both the substratum and the protein, as well as the time of addition of the enzyme relative to the conditioning of the substratum. The effects of dissolved and substratum-adsorbed proteins on the attachment of Pseudomonas sp. strain NCMB 2021 to petri dishes and tissue culture dishes were consistent with changes in bubble contact angles (except when proteins were adsorbed to tissue culture dishes before attachment) as were alterations in protein-induced inhibition of bacterial attachment to petri dishes by treatment with pronase. Differences between the attachment of pseudomonads to petri dishes and tissue culture dishes suggested that different mechanisms of adhesion are involved at the surfaces of these two substrata.     

Membrane fusion in the growth cone-microspike region of embryonic nerve cells undergoing axon elongation in cell culture

Fine structural analysis of embryonic nerve cells undergoing axon elongation in vitro has revealed structural evidence supportive of the time lapse cinematographic observation that extensive areas of active membrane fusion are present in the distal tip of the axon. Pre-fusion membrane alignment and post-fusion strings of vesicles characterize the putative fusions between microspikes, between microspikes and growth cones, and between growth cones and the distal axon. The restriction and possible significance of these apparent fusions are discussed.     

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Cell-substratum adhesion of neurite growth cones, and its role in neurite elongation.

The adhesion of chick embryo sensory neurons to glass coverslips was examined with interference reflection optics. On untreated glass, adhesive contacts are common only beneath growth cones and are small. On polylysine-treated glass growth cones are highly spread, microspikes reach treat lengths and extensive adhesive contacts underlie growth cones, microspikes and nerve fibers. Veils, expanded from the growth cone, are adherent to the substratum either centrally or laterally, while the extending edge of the cell margin is non-adherent. Linear adhesions are frequent beneath microspikes and pass centrally beneath the growth cone margin. The distribution of linear adhesions resembles that of microfilament bundles seen within whole mounts of growth cones. Adhesive contacts stabilize extensions of the growth cone margin and may influence the organization of the microfilamentous network within the growth cone. Regulation of microfilament organization by adhesion may influence microfilament functions in growth cone mobility and the assembly of neurite structures.     

Survival and development in culture of dissociated parasympathetic neurons from ciliary ganglia

Parasympathetic neurons from avian embryonic ciliary ganglia survive in low density culture when neurons are free from contact with other cells. A charged substratum, polyornithine, and a conditioned medium permit cell survival and vigorous neurite formation. The heart-conditioned medium must be present continuously and is active after dialysis. Neurites elongate rapidly, branch extensively, and follow patterns of charged substratum provided in the culture dish.     

Multiple differentiation of clonal teratocarcinoma stem cells following embryoid body formation in vitro

We have described the differentiation in vitro of clonal pluripotent teratocarcinoma stem cells derived from isolated single cells. By using solvent-resistant plastic petri dishes as a substratum for cell growth, it is possible to prepare histological sections of the cultures which can be compared with sections of teratocarcinomas formed in vivo by the same cells. Our results indicate that almost all of the cell types found in the tumors are formed in vitro, including cartilage, keratinizing epithelium, pigmented epithelium, neural tissue, and muscle. The cells are organized in a tissue structure which is remarkably similar to that found in vivo.     

Induction of hypoxia in glass versus Permanox petri dishes

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.     

Effects of cell adhesion to the substratum on the growth of chick embryo fibroblasts

Chick embryo fibroblasts were plated on Petri dishes that had not been treated for use in tissue culture (bacteriological dishes). On these dishes the cells grow at the same exponential rate as cells plated on tissue culture dishes, but their growth becomes inhibited sooner after plating, and therefore at a lower cell number per dish. The inhibition of cell growth on bacteriological dishes is correlated with the formation of cell clumps. Clump formation is reversible by mechanical transfer of the clumps to a tissue culture dish: the cells migrate out of the clumps, form a monolayer, and cell growth resumes.Clump formation was studied by time-lapse cinematography, and was found to be due to reduced adhesion of the cells to the bacteriological dish surface. This reduced adhesiveness of the substratum is due to a lower number of negatively-charged residues on the bacteriological dish surface, which can be measured by the binding of crystal violet. The number of negatively-charged residues, and therefore the adhesiveness of the substratum can be altered by treatment of the dishes with sulfuric acid. Serum components of the medium were found to affect cell adhesion to the bacteriological dishes, consequently altering the efficiency of cell attachment, the extent of cell growth and the pattern of clump formation.The cells in clumps were compared with those in confluent monolayers on tissue culture dishes. Growth-inhibited cells on both types of dish were found to be equally viable. Cells in clumps on bacteriological dishes were found to be inhibited in the G1 phase of the cell cycle, as are cells in density-inhibited monolayers. Infection by the oncogenic virus, Rous sarcoma virus, can release the cells from growth-inhibition on both types of dish. Cell-induced alterations of the medium are not involved in the growth inhibition of cells on bacteriological dishes.     

ULTRASTRUCTURE AND FUNCTION OF GROWTH CONES AND AXONS OF CULTURED NERVE CELLS

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells.

A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4 x 10(5) cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 microgram DNA or 5 micrograms protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

Improved methods for using glass coverslips in cell culture and electron microscopy.

For many applications, cells or tissue must be cultured on an optical surface of high quality. For such applications laboratories often prepare "special dishes," which are made by affixing a glass coverslip beneath a hole in a plastic petri dish bottom. In this report, we offer an improved method, using Parafilm as a dry mount adhesive, for the preparation of special dishes, and show that the resulting dish is non-toxic to neurons in culture. The Parafilm bond is stable at 60 degrees C, permitting electron microscopy resins to be poured directly into the dishes and cured. The glass coverslip can be readily removed from the cured resin mechanically. The techniques we describe offer time-saving and reliable improvements for the use of glass coverslips in cell culture and electron microscopy.     

Axon initiation by ciliary neurons in culture

A nerve culture system for the study of axon initiation is described. A population of individual chick embryo ciliary neurons, free from contact with other cells and attached to a polyornithinecoated culture dish, is exposed to heart cell-conditioned medium (HCM). Within 30 min after the addition of HCM the majority of neurons have formed growth cones, and by 90 min more than 80% of the neurons bear at least one axon longer than 15 μm. Before the addition of HCM, ciliary neurons generate membrane ruffles and extend filopodia around the entire periphery of the rounded cell body. Axon initiation, following addition of HCM, consists of two distinctive changes in the cell surface: (1) organization of the randomly distributed surface movements into localized highly active growth cones, which then form axons; and (2) the cessation of surface movements elsewhere on the cell periphery. Heart cell-conditioned medium may induce these changes by increasing the adhesion between parts of the nerve cell surface and the substratum.     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells

Summary A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4×10⁵ cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling, with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 μg DNA or 5 μg protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous, for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

Fibronectin and polylysine requirement for proliferation of neuroblastoma cells in defined medium

We have demonstrated in this study that we could eliminate the requirement of a serum preincubation for proliferation of B104 neuroblastoma cells in defined medium. When cells were plated directly into serum-free defined medium after trypsin or EGTA detachment, they had no difficulty in adhering or remaining attached to the plastic substratum but were incapable of cell division. However, the addition of human plasma fibronectin to serum-free defined medium and precoating the tissue culture dishes with polylysine at each subculture permitted cell division to occur. Fibronectin was only required at the time of subculture and did not need to be replenished at each medium change. In addition, we have shown that clonal growth and serial subculture are possible in serum-free defined medium provided that the cell inoculum encounters the appropriate substratum. These findings are consistent with a role for fibronectin and a positively charged substratum in the growth regulation of B104 neuroblastoma cells. This completely defined culture system will be of great benefit in studying the growth regulation and differentiation of these neuronal cells.     

Measurements of growth cone adhesion to culture surfaces by micromanipulation

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.     

Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II functions

Axon extension involves the coordinated regulation of the neuronal cytoskeleton. Actin filaments drive protrusion of filopodia and lamellipodia while microtubules invade the growth cone, thereby providing structural support for the nascent axon. Furthermore, in order for axons to extend the growth cone must attach to the substratum. Previous work indicates that myosin II activity inhibits the advance of microtubules into the periphery of growth cones, and myosin II has also been implicated in mediating integrin-dependent cell attachment. However, it is not clear how the functions of myosin II in regulating substratum attachment and microtubule advance are integrated during axon extension. We report that inhibition of myosin II function decreases the rate of axon extension on laminin, but surprisingly promotes extension rate on polylysine. The differential effects of myosin II inhibition on axon extension rate are attributable to myosin II having the primary function of mediating substratum attachment on laminin, but not on polylysine. Conversely, on polylysine the primary function of myosin II is to inhibit microtubule advance into growth cones. Thus, the substratum determines the role of myosin II in axon extension by controlling the functions of myosin II that contribute to extension.     

Characteristics of the cultivation of epidermocytes on collagen substratum

Epidermocytes obtained from human split-thickness skin were cultured in hypocalcium medium (ion calcium content was 0.14 mM). The half bottoms of culture plastic dishes were coated with collagen film before bringing cells in these dishes. It was proved that epidermocytes attach more quickly to collagen, than to plastic substratum, incorporate H3-thymidine (at early periods of cultivation) with larger rate and form and monolayer more quickly, than on the plastic substratum.     

The growth of spinal ganglion neurons in serum-free medium

The presence of serum in the culture medium affects the morphology of spinal ganglion neurons. With serum present, neuronal cell bodies are rounded and axons are predominantly straight. In serum-free medium axons curve all along their lengths, while both cell bodies and growth cones are spread on the substratum. Such “curved” axons straighten if serum is added to the culture dish.Serum-free medium may increase the adhesion of neurons to the substratum. This can account for the curved morphology of axons in serum-free medium, since such axons may represent a “history” of the movement of the growth cone during axon elongation.     

Variability of the Influence of Physicochemical Factors Affecting Bacterial Adhesion to Polystyrene Substrata

The role of electrostatic and hydrophobic interactions and solid and liquid surface tensions in the adhesion of four bacterial species (Pseudomonas fluorescens, Enterobacter cloacae, Chromobacterium sp., and Flexibacter sp.) to hydrophobic polystyrene petri dishes and to more hydrophilic polystyrene tissue culture dishes was investigated. The effect of electrostatic interactions was investigated by determining the effects of different electrolyte solutions on attachment to and of different electrolyte and pH solutions on detachment from the polystyrene substrate. The significance of solid and liquid surface tensions and hydrophobic interactions was investigated by measuring the effects of different surfactants (including a concentration series of dimethyl sulfoxide) on adhesion and detachment. Adhesion varied with bacterial species, substratum, and electrolyte type and concentration, with no apparent correlation between adhesion and electrolyte valence or concentration. The influence of different pH and detergent solutions on bacterial detachment also varied with species, substratum, pH, and detergent type; however, the greatest degree of detachment of all strains from the surfaces was produced by detergent treatment. The results suggest that adhesion cannot be attributed to any one type of adhesive interaction. There was some evidence for both electrostatic and hydrophobic interactions, but neither interaction could wholly account for the data.