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.
Highlights
S HAPE is a distinctive and sufficiently unique feature of cells to allow their identification and morphologic classification
The microfilamentous cytoskeleton of polymorphonuclear neutrophils (PMN) activated by chemotacticfactors or by substrate contact is constantly remodeled through actin polymerization and filamentous actin (F-actin) depolymerization [5, 27]
To determine whether F-actin distribution is critical for development of polar shape, we described the temporal relationship between F-actin distribution and change in shape of activated PMN
Summary
S HAPE is a distinctive and sufficiently unique feature of cells to allow their identification and morphologic classification. PMN change shape from round to polar, serially increase and decrease F-actin content, and apparently shift F-actin distribution from diffuse to focal [15]. These observations suggest that the amount and distribution of F-actin may play an important role in the development or maintenance of PMN shape. The evidence which supports this idea is circumstantial and derives primarily from fluorescence microscopy studies which localize F-actin to the subcortical region of the pseudopod of PMN or other cells [11, 15, 22]. No study of cytoskeleton and shape uses a round cell which can assume any shape to directly examine the relationship between temporal change in microfilamentous cytoskeleton organization and acquisition of shape
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