Joan E.M. Heaysman’s research while affiliated with University College London and other places

The attachment under gravity to a glass surface and subsequent spreading of chick heart fibroblasts has been studied by phase contrast, reflection interference (RIM), scanning electron (SEM) and transmission electron microscopy (TEM). It has been shown that a cell can pass from the rounded state to a fully polarized locomotory state in as short a time as 5 min. The initial stable attachment of the cell to the substratum is brought about by small-diameter protrusions. Fully polarized cells already display areas of close apposition to the substratum with sub-membranous filamentous aggregations analogous to the ‘plaques’ described previously in locomoting fibroblasts.

Reflection interference microscopy was used to demonstrate that reducing the electrolyte concentration of the medium (sucrose was added to the medium to keep it isotonic) resulted in large areas of the ventral surface of the avian fibroblast moving further away from the substrate. Thus in cells 3 h after plating out the grey reflection image changed to white and in cells 24 h after plating out the predominantly white image was lost as the ventral surface moved even further away. These changes were reversible on replacing the low ionic strength solutions with 199 medium or 145 mM NaCl. This offers direct experimental evidence for the role of long range electrostatic forces in cell-substrate interactions of fibroblasts.

This chapter discusses the contact inhibition of locomotion. Contact inhibition is defined as the stopping of the continued locomotion of a cell in the direction which has produced a collision with another cell or—alternatively, the prohibition when contact between cells has occurred—of continued movement such as would carry one cell across the surface of another. The original data on contact inhibition of locomotion were obtained from studies on the displacement of the whole cell. Explants were placed so that the cellular outgrowths met between them. The formation of a more-or-less single layer of cells over the substrate and the virtual absence of nuclear overlaps rapidly came to be used as criteria for the presence of contact inhibition, while a high incidence of nuclear overlap, multilayering, or piling up, was considered evidence of its absence. The amount of overlap in a particular situation was expressed as an overlap index. This value was derived by estimating the number of cell nuclei which would be expected to overlap if the cells were distributed at random and comparing this number with the number of overlaps actually occurring, the ratio of the observed number to the expected number being the index.

When chick heart fibroblasts collide with zinc-fixed fibroblasts in culture, they show a typical ‘contact inhibition of locomotion’ reaction both an inhibition of overlapping and contact paralysis occurring. Previous reports in the literature have suggested that contact inhibition of locomotion only occurs when the collision is between living cells.

Electron microscope observations of cells from methylcholanthrene induced tumours of the mouse and mouse muscle fibroblasts, grown in vitro, show five main differences in ultrastructure. The surface of the normal fibroblast is smooth when compared with that of the tumour cell; the leading lamella of the normal fibroblast has fewer ruffles (lamellipodia) and fewer cell-substrate plaques than the tumour cell; the tumour cell does not have a layer of cortical filaments like the normal cell but has well marked filamentous tracts in the main body of the cytoplasm and the endoplasmic reticulum of the normal cell is far less abundant and wellorganized than that of the malignant cell. The fact that all these differences are possibly related to the cell surface may be of significance when considering the changes brought about during carcinogenesis.

When chick heart fibroblasts and mouse sarcoma (S180) cells make contact with each other, an ultrastructural study shows no formation of cortical specialisations at the area of contact, as are seen in the case of fibroblast-fibroblast contacts.

Pairs of chick heart fibroblasts have been studied with the light microscope and then fixed in situ for electron microscopy at varying times after they have been seen to make contact with each other. The resultant electron micrographs show that areas of specialisation begin to develop within 20 sec of the contact being made. These specialisations resemble those seen in isolated chick heart fibroblasts where the cell comes close to the substrate and are thought to be areas of adhesion. The development of these areas and their associated microfilaments is described and an attempt is made to correlate this with the stages of the contact inhibition phenomenon.

Pairs of chick heart fibroblasts have been studied with the light microscope and then fixed in situ for electron microscopy at varying times after they have been seen to make contact with each other. The resultant electron micrographs show that areas of specialisation begin to develop within 20 sec of the contact being made. These specialisations resemble those seen in isolated chick heart fibroblasts where the cell comes close to the substrate and are thought to be areas of adhesion. The development of these areas and their associated microfilaments is described and an attempt is made to correlate this with the stages of the contact inhibition phenomenon.

When fibroblasts that have had their surfaces marked with concanavalin A at room temperature are allowed to resume movement, the marker is rapidly cleared from the anterior end of the cell. This supports the indication from marking with particles that new surface is generated anteriorly during fibroblast movement.

When fibroblasts that have had their surfaces marked with concanavalin A at room temperature are allowed to resume movement, the marker is rapidly cleared from the anterior end of the cell. This supports the indication from marking with particles that new surface is generated anteriorly during fibroblast movement.