Involvement of Membrane Skeletal Proteins in the Stimulus-Response Coupling of the Neutrophil and Its Molecular Mechanism

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1. By preembedding immunoelectron microscopy, most(>75%)of fodrin in the normal human neutrophil was not seen on the cell membrane nor within 100 nm from it, but in the filamenlous cytoplasm further inside. This result was distinct from those with speclrin in the erythrocyte and fodrin in the lymphocyte, in which a majority of immunogold labeling was attached to the cell membrane. The result indicates that fodrin in the neutrophil is a cytoskeletal protein rather than a peripheral membrane Drotein.2. Neutrophils treated with a chemotactic peptide, f-Met-Leu-Phe, became morphologically differential"led along the front-tail axis, and fodrin became concentrated in the posterior region ; the percentage of cells showing the polarized distribution of fodrin was more than 70% at 2 min, reached the maximum of around 80% at 10 min at room temperature, and decreased thereafter. The drastic redistribution occurs much faster than the similar relocation of fodrin in capping lymphocytes, which generally takes more than 10 min at 37ﾟC.3. The superior cell membrane of adherent neutrophils was removed and the remaining basal half of the cells was labeled for immunogold electron microscopy(unroofing method). In unstimulated cells, immunolabeling was observed uniformly in the membrane and filaments of 5 nm in diameter were labeled occasionally ; after stimulation with the chemotactic peptide, labeling was concentrated in the posterior half of the cell where numerous bundles of 10 nm filaments run in parallel. The result indicates that fodrin mediates'the redistribution of cytoskeletal components in the neutrophil.4. Similar dynamic redistribution was observed in the epidermis and the corneal epithelium. The unroofing method was soplied to the chromaffin cell and filaments of 2-3 nm in diameter were found labeled for fodrin.