VISUALIZATION OF ACTIN-FILAMENTS IN KERATOCYTE LAMELLIPODIA - NEGATIVE STAINING COMPARED WITH FREEZE-DRYING

Depending an the method of preparation, the actin-rich lamellipodia of motile cells can show very different structural organizations. This situation has been a main contributor to differences in current ideas about the possible mechanisms of cell movement. We have here analyzed the structure of the lamellipodium in whole-mount cytoskeletons using one of the most rapid of crawling cells, the fish keratocyte, employing two procedures considered least damaging to actin filament arrays: freeze-drying and negative staining. At the front of the lamellipodium, where filaments density is the highest, freeze-dried images conveyed the impression of a cross-linked network of very short, interconnected filaments-as previously observed by others-whereas the same regions appeared as a diagonal meshwork of long, more or less straight filaments after negative staining. In general, the linearity of actin filaments was not preserved after freeze-drying, except in situations where the filaments had partially dried down onto the substrate before freezing. In the mid and posterior regions of the lamellipodium the actin filaments appeared to be up to several micrometers long by negative staining, whereas their length was impossible to discern by freeze-drying, owing to filament kinking and aggregation and to the nature of the contrasting procedure, which reveals only the upper layers of filaments. We conclude that while freeze-drying preserves the overall three-dimensional structure of the lamellipodium it also introduces fine-structural distortions in actin that obscure actin filament order. Drying in negative stain appears to stabilise the actin network. Details of associated crosslinking structures are lost in both techniques. The data indicate the importance of adopting independent, complementary and ideally new approaches for elucidating the organization of the actin cytoskeleton. (C) 1994 Academic Press, Inc.