DISTRIBUTION OF MYOSIN AND ACTIN IN MOVING HUMAN NEUTROPHILS DETECTEDBY DOUBLE-FLUORESCENCE STAINING

OBJECTIVE: To observe the distribution of myosin and actin in the same phase in the same human neutrophils with a double-fluorescence staini ng procedure that readily evaluates the association of myosin and acti n in human neutrophils during movement. STUDY DESIGN: Contractile prot eins, consisting mainly of myosin and actin, are essential to cell mot ility. Motile forms of human neutrophils were fixed in formaldehyde an d glutaraldehyde and stained for fluorescence analysis. The prepared s lide was first observed with a green filter for fluorescein isothiocya nate-labeled antimyosin, with a red filter used for rhodamine-labeled phalloidin and without a filter to obtain a phase contrast image. RESU LTS: This method made it possible to clearly demonstrate the localizat ion of myosin in the cytoplasm. The localization of myosin differed fr om that of actin. The pattern of distribution suggested a close associ ation between myosin and actin. Actin was reorganized mostly in the fi lopodia, and myosin was organized in the peri-granular area. CONCLUSIO N: Our method utilizes conventional fluorescence microscopy, rather th an confocal fluorescence microscopy, and was useful for investigating the shifts in localization of myosin and actin in the motile forms of such small cells as human neutrophils.