title: Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils.
abstract: Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN were labeled with fluorescent probe NBD-phallicidin to measure filamentous actin and Texas red to assess cell thickness. We show that Texas red fluorescence is a reasonable measure of cell thickness and that correction of the NBD-phallicidin image for cell thickness using the Texas red image permits assessment of focal F-actin content. Parameters were derived that quantify total F-actin content, movement of F-actin away from the center of the cell, asymmetry of F-actin distribution, and change from round to polar shape. The sequence of change in F-actin distribution and its relation to development of polar shape in PMN was determined using these parameters. After stimulation with chemotactic peptide at 25 degrees C, F-actin polymerized first at the rim of the PMN. This was followed by development of asymmetry of F-actin distribution and change to polar shape. The dominant pseudopod developed first in the region of lower F-actin concentration followed later by polymerization of actin in the end of the developed pseudopod. Asymmetric F-actin distribution was detected in round PMN before development of polar shape. Based upon these data, asymmetric distribution of F-actin is coincident with and probably precedes development of polar shape in PMN stimulated in suspension by chemotactic peptide.
authors.affiliation: Division of Pediatric Hematology/Oncology, Childrens Hospital Los Angeles, University of Southern California.
authors.forename: T D
authors.initials: TD
authors.lastname: Coates
authors.forename: R G
authors.initials: RG
authors.lastname: Watts
authors.forename: R
authors.initials: R
authors.lastname: Hartman
authors.forename: T H
authors.initials: TH
authors.lastname: Howard
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000199:Actins / Q000648:ultrastructure*; D002634:Chemotaxis, Leukocyte; D006801:Humans; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology*; D010590:Phalloidine; D013997:Time Factors; D014741:Video Recording
chemical_list: D000199:Actins; D010590:Phalloidine; D009240:N-Formylmethionine Leucyl-Phenylalanine