The Effect of Neuraminidase (3:2:1:18) on the Aggregation of Cells Dissociated from Embryonic Chick Muscle Tissue

Abstract

Embryonic chick muscle cells were used to investigate the effect of removing cell-surface sialic acids on cell aggregation in vitro. Single cell suspensions were prepared by dissociating skeletal muscle tissue of 9-day-old chick embryos with either crystalline or crude trypsin. Cell aggregation was quantitatively estimated by turbidimetric and gyratory shaker methods. Cells dissociated with crude trypsin and suspended in Hanks's balanced salts solution (BSS) containing 25u./ml neuraminidase (NANase) only aggregated for 2h when rotated in an absorptiometer. The inhibitory effect of the enzyme was more pronounced with increasing concentration up to 25u./ml. Cells dissociated with crystalline trypsin and treated with 100u./ml NANase immediately exhibited a reduced aggregative competence when gyrated in Eagle's minimum essential medium (MEM) containing 25u./ml NANase, compared with the controls which were not exposed to NANase. The aggregation rate of muscle cells pretreated with 100u./ml NANase and suspended in Eagle's MEM was similar to that of the untreated controls. Cell counts showed that under all three experimental conditions cells were not added to aggregates after the 12-h stage. Aggregates formed in Eagle's MEM (the controls) joined together to form larger aggregates after 12 h, but those rotating in the presence of NANase did not display this property. Lissamine green viability tests showed that cells remained alive throughout the 24-h period in the presence of NANase. Determinations of oxygen uptake, protein synthesis and mitotic index confirmed that general cellular viability was not affected by NANase. Fluorescent-labelled NANase was not taken up by the cells. Treatment of crystalline trypsin-dissociated muscle cells with 100u./ml NANase for 30 min at 37°C significantly reduced their negative electrophoretic mobility. This diminution closely corresponded to the removal of cell-surface sialic acids, as measured by colorimetric tests. Interpretation of the results in the light of current theories of cell adhesion failed to give support to the concept of adhesion by physical forces. The mechanism by which cellular deformability could influence cellular adhesiveness is modified in the knowledge of the present results.