Reorganization of cytoskeletal and contractile elements during transition of human monocytes into adherent...

Indirect immunofluorescence microscopy was used to study the reorganization of contractile and cytoskeletal elements of human monocytes during their in vitro transition into macrophage-like cells. In freshly isolated monocytes, actin, myosin, alpha-actinin, and vinculin all had a diffuse distribution, whereas microtubules and vimentin-type intermediate filaments showed a distinct fibrillar organization. In culture, the cells differentiated within 1 week to two distinct cell types: bipolar fibroblastoid cells and flattened epithelioid cells. A conspicuous redistribution of actin, alpha-actinin, and vinculin from a diffuse cytoplasmic location into distinct punctate foci at the substratum-facing side of the cells was seen already at the early attachment phase of the spreading monocytes. In spread cells the punctate foci occupied the whole ventral surface of the cells, and in flattened, fully spread cells these proteins formed distinct punctate plaques at the under surface of the cells excluding however, the ruffle edge-like membrane regions. At the plaques, a close co-distribution of actin and vinculin was seen in double indirect immunofluorescence microscopy. Myosin showed a distinctly different reorganization during the transition process changing from a diffuse cytoplasmic location to a striated, surface-associated distribution. Interference reflection microscopy revealed large areas of close adhesion but no focal adhesion sites in spreading monocytes. Cytochalasin B treatment lead to a rapid distortion of the actin organization and resulted in clump-like cytoplasmic aggregates and nuclear paracrystals of actin concomitantly with the rounding up of the cells. Vimentin filaments were seen as a perinuclear aggregate until the acquisition of the fully spread morphology. At this stage both vimentin filaments and microtubules displayed a fibrillar appearance throughout the cytoplasmic domain. Treatment of the spread monocytes with antimitotic drugs caused a retraction of the cell from beyond the attachment plaques but had no effect on the punctate actin organization. The results show that in the monocyte-macrophage transition there is an extensive cytoskeletal reorganization that can be correlated with the different phases of the cell-to-substratum attachment process.