Fig. 7. CMS and CIN 85 crosslink individual F-actin filaments into bundles. (A) Biochemical actin bundling assay. Freshly polymerized non-muscle actin was incubated for 30 minutes with -actinin, GST and CMS CT (left panel), or with GST, CMS CT, CT PR, CT CC and CIN85 CT. The F-actin bundles were separated from soluble F-actin filaments by centrifugation, and the pellets were subjected to western blot analysis. CMS CT and CIN85 CT caused actin pelleting, with a detectable depletion of the actin amount in the CMS CT supernatant. (B) TEM of negatively stained preparations of actin filaments and F-actin crosslinked by CMS CT and -actinin. F-actin bundles formed when freshly polymerized, non-muscle F-actin was incubated with CMS CT, CIN85CT or -actinin (positive control). The CC domain is crucial for the formation of actin filament crosslinks, because in the presence of CMS CC no bundles were formed similarly to GST (negative control). Bars, 100 nm. (C) These findings allow us to propose a model for the interaction of CMS with F-actin. The CC domain plays a dual role: being important in actin binding and filament crosslinking.