Journal of Cell Science, Vol 16, 385-399, Copyright © 1974 by Company of Biologists

Submitted on March 26, 1974

Limpet Haemocytes

III. Effects of Cytochalasin B and Colchicine on Cell Spreading and Aggregation

¹ Biology Department, Queen Elizabeth College, London W8, England
² Department of Experimental Pathology, Charing Cross Hospital Medical School, London W6, England

Limpet blood cells are suitable for the study of a number of interdependent features of cell behaviour; microspike formation, spreading and locomotion on a solid substrate, and aggregation. The effects of cytochalasin B (CCB) and colchicine on each of these activities is studied with a view (a) to determining with which functions these 2 reagents interfere; and (b) to gaining information on the relationships between these functions in normal cell behaviour.

Rapid spike formation and fast aggregation in a shaker system, which are normal features of the behaviour of amoebocytes in limpet blood or in seawater, are totally but reversibly inhibited by the presence of low concentrations of CCB (e.g. 0.5 µg/cm³). Similarly, the spreading of amoebocytes on to a solid substrate is greatly inhibited by CCB, both in rate and extent, and the rapid locomotion on a solid substrate of normal amoebocytes is completely abolished by CCB. The first observable effects of CCB on spread amoebocytes are loss of optical integrity of the spikes and retraction of parts of the anterior cell margin. The loss of distinctive cell shape, the inhibition of spreading on to a glass surface and the lack of motility on such a surface may all be immediate consequences of the disruption of spike structure.

CCB inhibits aggregation but does not disrupt preformed contacts between cells, which suggests that it acts on an early stage in the formation of stable contact rather than on the adhesiveness per unit area of contacting cell surface. A possible link between the effects of CCB on spikes and on aggregation is the proposal that low-diameter projections are needed to establish initial contact between cells. Alternatively, the spikes may be required for the rapid spreading of cells, not only on to glass but also over the surfaces of other cells, enabling them to increase their mutual contact area very rapidly and thus stabilize an adhesion.

Amoebocytes maintained in the presence of 50 µg/cm³ colchicine for a number of hours gradually lose their bipolar form and the entire cell margin becomes occupied by the spike-supported lamella which normally constitutes the leading edge of the cell. Thus microtubules are probably not necessary for the skeletal functions of spikes or for their roles in spreading and aggregation, but they do appear to play a part in the control of spike orientation.

Macrophages, cells lacking obvious spikes and showing little sign of bipolarity, appear unaffected by CCB or colchicine and spread normally on to a glass slide in the presence of either reagent. This, together with the limited spreading of amoebocytes in CCB, suggests that at least 2 distinct mechanisms may operate in the spreading of cells on to a solid substrate.