Fig. 4. (A) Colocalization of -actinin with F-actin in microspikes during IGF-I-induced cell separation. MCF-7/IGF-IR/WT cells, untreated (a-c) or stimulated with 50 ng/ml IGF-I for 5 minutes (d-f), were fixed in 3.7% formaldehyde and co-stained for F-actin with TRITC-phalloidin and for -actinin with a monoclonal antibody to -actinin. The images of middle sections acquired by confocal laser-scanning microscopy illustrate staining for F-actin in red (a,d) and -actinin in green (b,e). In overlays (c,f), sites of -actinin and F-actin colocalization appear in yellow. Insets in d-f: examples of F-actin and -actinin-containing microspikes from the zone of the intercellular membrane ruffles pointed to by arrow (d-f). Bar, 10 µm. (B) Ultra-structural analysis of IGF-I-induced microspikes in MCF-7/IGF-IR/WT cells. (a) Examples of adherens junction (aj), desmosome (d) and intermediate filaments (if) proximate to desmosomes are indicated in confluent serum-starved cells. The longitudinal section of continuous contacts between lateral membranes is shown in the upper inset. (b) Cross-sections through microspikes (MS) formed in the cells stimulated with 50 ng/ml IGF-I for 15 minutes. Individual bundles of actin in the core of MS formed by opposing cells are pointed out by a row of arrows; gaps between single microspikes are labeled with asterisks. A membrane site resembling an immature or disassembling desmosome in close proximity to intermediate filaments is labeled d. In the inset, the horizontal section through apical zones of IGF-I-stimulated cells is shown. N, nucleus. Bar, 250 nm. In insets, bar, 5 µm.