Use of the ADP-ribosyltransferase C3 from Clostridium botulinum resulted in the identification of the involvement of the Rho proteins in the regulation of the microfilament system. The advantage of C3 (selective modification of RhoA, B and C) is offset by the disadvantage of poor cell accessibility. In contrast, toxin B can enter the cells by a specific mechanism. Therefore, the concentration needed is quite low and the incubation times are in a moderate range (2-6 h). However, toxin B glucosylates not only Rho subtype proteins, but also all members of the Rho subfamily (Rho, Rac and Cdc42). Although these GTPases are involved in the control of the actin cytoskeleton, each of them exhibits a specialized function in the regulation of the complex microfilament system. Furthermore, these Rho GTPases exhibit functions which are apparently not related to the cytoskeleton; for example, they participate in the regulation of transcription factors (Minden etai, 1995; Hill etal., 1995; Coso etal., 1995; Olson etal., 1995), and control exocytosis of transmitters in rat basophilic leukemia cells (Pre-pens etal., 1996).
Jk It is therefore necessary to distinguish between cellular effects
<yj> caused merely by depolymerization of the actin filaments and effects caused by inactivation of the Rho GTPases which are independent of comparison with C2 toxin the actin system. To this end, it is very helpful to compare effects of toxin B with those of Clostridium botulinum C2 toxin. C2 toxin directly acts on the cellular G-actin by ADP-ribosylating actin at Arg-177. This modification blocks the ability of actin to polymerize, and makes it a capping protein. Both mechanisms eventually result in almost complete depolymerization of the cellular actin cytoskeleton (for review see references Aktories and Just, 1990; Aktories et a/., 1992; Aktories and Wegner, 1992; and see Chapters 8 and 11). By using C2 toxin, the direct effects of the actin cytoskeleton on the functions studied can be monitored.
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