F-actin distribution in polymorphonuclear leucocytes

The polarization and amoeboid locomotion of neutrophil leucocytes is stimulated by chemotactic factors, which initiate waves of contraction in both adherent and non-adherent neutrophils. These cyclical contractile events have previously been analysed by time-lapse filming but the mechanisms involved in the coordination of the cytoskeleton during locomotion have not been elucidated, one reason being because of the problems involved in fixing motile cells. In this paper we show that improved fixation of motile neutrophils with low concentrations of glutaraldehyde followed by glycine quenching demonstrated significant differences in the pattern of staining with TRITC-phalloidin in neutrophils moving on different substrata. Previous film analysis had shown the basic features of locomotion to be similar on all substrata. A prominent feature of leucocyte locomotion on two-dimensional substrata (e.g. protein-coated glass), on three-dimensional collagen gels or in motile cells floating in suspension, is the wave of contraction that passes antero-posteriorly along the length of the cell. The organization of the cytoskeletal elements has not been demonstrated at contraction waves, but light fixation with glutaraldehyde followed by staining with TRITC-phalloidin demonstrated prominent bands of F-actin in neutrophils inside collagen gels. These bands were not present in neutrophils either in suspension or moving on a two-dimensional substratum. Although all motile neutrophils had brightly stained anterior lamellipodia, the cells moving on the two-dimensional substratum had very extensively ruffled leading lamellae stained very brightly with TRITC-phalloidin. The reasons for the absence of consistent bands of F-actin at contraction waves are discussed.