the early stage of differentiation is crucial for unimpeded osteoclast-formation and imply that the C3-catalyzed inhibition of Rho-signalling only at day 0 is sufficient to inhibit osteoclastformation. The effect of C3-treatment on fully differentiated osteoclasts regarding their resorption activity was investigated. RAW 264.7-derived osteoclasts were grown on synthetic calcium phosphate surfaces and treated with C2IN-C3lim from day 5 on, i.e. after the formation of multi-nucleated osteoclasts, to clearly distinguish between toxin effects on osteoclastformation and activity. Quantification of the resorbed areas after 13 days of Telepathine culture revealed a significantly reduced resorption by osteoclasts after C3-treatment. Again, treatment of the cells with enzymatically inactive C3bot1E174Q had no effect on the resorption by osteoclasts, indicating that the C3-catalyzed Rho-modification in the cytosol of the osteoclasts was crucial for this effect. Finally, the uptake of C2IN-C3lim into differentiating osteoclasts and the morphological changes induced by C3-treatment were analyzed by immunofluorescence microscopy. RAW 264.7 cells cultured in the absence of C2IN-C3lim with RANKL formed cells with multiple nuclei. Moreover, their actin cytoskeleton was organised in a seemingly ring-like structure as mainly anticipated for potentially active osteoclasts. Cells cultured in the presence of C2IN-C3lim showed a characteristic change of their morphology including shrinking of the cell bodies and formation of multiple thin protrusions accompanied by re-organization of the actin cytoskeleton, which is characteristic for C3-treated cells. The typical morphological changes indicate that enzymatically active C3 was 1439901-97-9 present in the cytosol of these cells. The detection of cell-associated C2IN-C3lim with specific C3-antiserum confirmed the C3-uptake into differentiating osteoclasts, whereas no C3-specific staining was detected in untreated control cells. The images of C2IN-C3lim-
