Distribution of myosin and actin in moving human neutrophils detected by 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 staining procedure that readily evaluates the association of myosin and actin in human neutrophils during movement.


METHODS
Contractile proteins, consisting mainly of myosin and actin, are essential to cell motility. Motile forms of human neutrophils were fixed in formaldehyde and glutaraldehyde and stained for fluorescence analysis. The prepared slide was first observed with a green filter for fluorescein isothiocyanate-labeled antimyosin, with a red filter used for rhodamine-labeled phalloidin and without a filter to obtain a phase contrast image.


RESULTS
This method made it possible to clearly demonstrate the localization of myosin in the cytoplasm. The localization of myosin differed from that of actin. The pattern of distribution suggested a close association between myosin and actin. Actin was reorganized mostly in the filopodia, and myosin was organized in the perigranular area.


CONCLUSIONS
Our method utilizes conventional fluorescence microscopy, rather than 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.
Takubo
T
T
Department of Clinical and Laboratory Medicine, Osaka City University Medical School, Japan.
Tatsumi
N
N
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D002465:Cell Movement; D006801:Humans; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D000199:Actins; D009218:Myosins