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 s ituation has been a main contributor to differences in current ideas a bout the possible mechanisms of cell movement. We have here analyzed t he structure of the lamellipodium in whole-mount cytoskeletons using o ne of the most rapid of crawling cells, the fish keratocyte, employing two procedures considered least damaging to actin filament arrays: fr eeze-drying and negative staining. At the front of the lamellipodium, where filaments density is the highest, freeze-dried images conveyed t he impression of a cross-linked network of very short, interconnected filaments-as previously observed by others-whereas the same regions ap peared 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 fila ments appeared to be up to several micrometers long by negative staini ng, whereas their length was impossible to discern by freeze-drying, o wing to filament kinking and aggregation and to the nature of the cont rasting procedure, which reveals only the upper layers of filaments. W e conclude that while freeze-drying preserves the overall three-dimens ional structure of the lamellipodium it also introduces fine-structura l distortions in actin that obscure actin filament order. Drying in ne gative stain appears to stabilise the actin network. Details of associ ated crosslinking structures are lost in both techniques. The data ind icate the importance of adopting independent, complementary and ideall y new approaches for elucidating the organization of the actin cytoske leton. (C) 1994 Academic Press, Inc.