Immunoelectron microscopy of fodrin in the unroofed speciment of the adherent human neutrophil.

Abstract

We reported previously that fodrin is concentrated in the posterior portion of the human neutrophil stimulated with the chemotactic peptide (Fujimoto and Ogawa, J. Cell Sci., 96: 477-486, 1990). In the present study, the mechanically unroofed specimen of glass-adherent neutrophils was examined to define finer details of fodrin distribution. By immunofluorescence microscopy, F-actin and fodrin were positively labeled; after stimulation with the chemotactic peptide, fodrin was found more densely in the posterior half of the cell as in the whole cell observation. In ultrathin sections of the unroofed and immunogold-labeled specimen, two dimensional distribution of fodrin could be observed. Some gold particles were observed with filaments of about 5 nm in diameter, but others were on amorphous materials. In the stimulated neutrophil, labeling for fodrin was mainly found between straight filaments of about 10 nm in diameter which were densely distributed in the posterior portion of the cell. The results indicate that the redistribution of fodrin in the stimulated neutrophil is concurrent with other cytoskeletal changes.Cell migration is enabled by a combination of multiple factors, but obviously cytoskeleton is the major player in the phenomenon (17). In moving cells, various cytoskeletal proteins show redistribution, and accumulation either in the anterior or in the posterior portion has been reported: F-actin (10, 14), myosin II and actin-binding protein (18) in neutrophils and myosin I in amoebae (9) are seen anteriorly, whereas myosin II in amoebae is in the posterior (9).In the previous study, we observed that fodrin is concentrated in the posterior portion of migrating neutrophils stimulated with the chemotactic peptide (8). Moreover, fodrin was not localized to the cell membrane itself, but in the peripheral cytoplasm appearing highly filamentous in thin sections. We therefore concluded that fodrin is a cytoskeletal rather than a peripheral membrane protein and may be related to the dynamic reorganization of the cytoskeleton in the neutrophil. A question raised by the result was where and how fodrin is localized in the macromolecular architecture of the cell. Presently we utilized a cytochemical technique which allows distinct visualization of the membrane associated cytoskeletal components, and localized fodrin in direct correlation with them.