Double localization of F-actin in chemoattractant-stimulated polymorphonuclear leucocytes.
Uniform concentrations of chemoattractants such as formylpeptides induced a morphological polarization of human polymorphonuclear leucocytes (PMNs) and a concentration of F-actin at the cell front. They also induced a transient increase in filamentous actin (F-actin) which preceded the cell shape change. We combined fluorescence microscopy and image analysis to study the localization of F-actin, as revealed by a specific probe (bodipyTM phallacidin) in suspended PMNs stimulated by chemoattractants. F-actin exhibited remarkable concentration in focal points after a 30 s exposure to 10(-8) M formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), although no shape change of PMNs was detectable. A 10-min incubation with formylpeptide (10(-6) to 10(-9) M) induced the morphological polarization of PMNs and the appearance of a principal focus of F-actin in the cell head region and a secondary focus in the cell posterior end. The distribution of F-actin-associated fluorescence in 2D images of polarized PMNs might be due to an actual concentration of F-actin in privileged areas, to a local concentration of plasma membrane drawing filamentous actin or to variations in the cell volume. Then, we studied the distribution of a cytoplasmic marker, fluorescein diacetate and a membrane probe, TMA-DPH, in unstimulated rounded PMNs and in spherical and morphologically polarized PMNs stimulated by formylpeptide. The distribution of neither of these probes was correlated with F-actin distribution, especially in rounded PMNs stimulated 30 s with 10(-8) M fMet-Leu-Phe, suggesting that F-actin was concentrated in two foci located in the cell head region and in the cell posterior end. In addition, zymosan-activated serum induced the morphological polarization of PMNs and the appearance of two foci of filamentous actin, demonstrating that binding of formylpeptide to its specific receptor was not required for F-actin reorganization. We conclude that the accumulation of F-actin probably resulted from local filament assembly and put forward the hypothesis that microfilament reorganization in two centres drives the morphological polarization of PMNs.
Lepidi
H
H
Laboratoire d'Immunologie, Hôpital de Sainte-Marguerite, Marseille, France.
Benoliel
A M
AM
Mege
J L
JL
Bongrand
P
P
Capo
C
C
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000328:Adult; D016764:Cell Polarity / Q000187:drug effects; D005456:Fluorescent Dyes; D006651:Histocytochemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*
D000199:Actins; D005456:Fluorescent Dyes; D009240:N-Formylmethionine Leucyl-Phenylalanine