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Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils
T D Coates1, R G Watts, R Hartman
1Division of Pediatric Hematology/Oncology, Childrens Hospital Los Angeles, University of Southern California.
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.
Insights
Human neutrophils (PMN) change shape by polymerizing filamentous actin (F-actin). Asymmetric F-actin distribution precedes and coincides with the development of polar shape in PMN after stimulation.
Area of Science:
- Cell Biology
- Biophysics
Background:
- Actin polymerization is crucial for cell shape changes in human neutrophils (PMN).
- Understanding filamentous actin (F-actin) distribution is key to elucidating PMN polarization.
Purpose of the Study:
- To develop and validate a method for quantifying F-actin distribution in single PMN.
- To investigate the relationship between F-actin distribution and shape change in stimulated PMN.
Main Methods:
- Developed computerized video image analysis and fluorescence microscopy.
- Used NBD-phallicidin for F-actin and Texas red for cell thickness.
- Corrected F-actin images for cell thickness to assess focal F-actin content.
Main Results:
- F-actin initially polymerized at the cell rim upon stimulation.
- Asymmetric F-actin distribution preceded polar shape development.
- Pseudopod formation occurred in regions of lower F-actin concentration, followed by actin polymerization.
Conclusions:
- Asymmetric F-actin distribution is a key event preceding and accompanying PMN polar shape development.
- The developed method allows for quantitative assessment of F-actin dynamics and cell shape changes.
- This study provides insights into the spatiotemporal regulation of actin polymerization during neutrophil polarization.