Relationship of F-Actin Distribution to Development of Polar Shape in Human Polymorphonuclear Neutrophils

• Published in: The Journal of cell biology 1992-05, Vol.117 (4), p.765-774
• Main Authors: Coates, Thomas D., Watts, Raymond G., Hartman, Raymond, Howard, Thomas H.
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - ultrastructure; Actins; Actins - ultrastructure; Biological and medical sciences; Cell Biology; Cell physiology; Chemotaxis, Leukocyte; Cytoskeleton; Dot product of vectors; Fluorescence; Fundamental and applied biological sciences. Psychology; Geometric centers; Humans; In Vitro Techniques; Kinetics; Life Sciences & Biomedicine; Microscopy; Microscopy, Fluorescence; Molecular and cellular biology; Motility and taxis; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Neutrophils; Neutrophils - cytology; Phalloidine; Polymerization; Ratios; Science & Technology; Time Factors
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.117.4.765

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°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.