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SummaryThe binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.

Amplification and overexpression of the receptor tyrosine kinase ErbB2 occur in up to 30% of human breast cancers, and high ErbB2 levels are correlated with poor prognosis for breast cancer patients. In contrast to the epithelial growth factor receptor (ErbB1), ErbB2 is not downregulated by ligand-induced mechanisms. Here we show that flotillins are involved in the stabilization of ErbB2 at the plasma membrane. In SKBR3 breast cancer cells and breast cancer tissue, a positive correlation between flotillin and ErbB2 expression levels could be demonstrated. Moreover, the tissue microarray analyses of biopsies from 194 patients diagnosed with carcinomas of the breast showed that flotillin-2 emerged as a potential predictor of prognosis in breast cancer. Depletion of flotillin-1 and flotillin-2 leads to internalization and degradation of ErbB2. Furthermore, flotillin-1 and -2 were found to be in a molecular complex with ErbB2 and Hsp90. The depletion of one of these proteins results in disruption of this complex, followed by destabilization of ErbB2 at the membrane, and its internalization and degradation. As a consequence, ErbB2-triggered downstream signalling is inhibited. Our data demonstrate a novel mechanism for interfering with ErbB2 signalling, which potentially can have clinical impact.

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membranecytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.

The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.Clostridium botulinum C2 toxin, the prototype of the family of binary actin ADP-ribosylating toxins, mono-ADP-ribosylates G-actin at Arg-177 (1). C2 toxin consists of the enzyme component C2I (431 amino acid residues, 49.3 kDa) and the binding/translocation component C2II (721 amino acid residues, 80.8 kDa). The ADP-ribosyltransferase activity of C2I is located in its C-terminal domain (5), while the enzymatically inactive N-terminal domain (C2IN, amino acid residues 1 to 225) mediates interaction with C2IIa and translocation of C2I into the cytosol (6). Following limited proteolysis, the active C2IIa protein (ϳ60 kDa) forms ring-shaped heptamers that assemble with C2I and mediate binding of the toxin complex to the cellular receptor, a carbohydrate structure (2, 11, 19). During cellular uptake, the heptamers form pores in endosomal membranes, thereby facilitating translocation of C2I into the cytosol (7). Translocation of C2I requires unfolding (14) and subsequent refolding of the protein in the cytosol by the host cell chaperone Hsp90 (13).In the cytosol, C2I catalyzes covalent transfer of the ADPribose moiety from NAD (NAD ϩ ) to Arg-177 of G-actin (1). Mono-ADP-ribosylation of actin induces the depolymerization of F-actin and thereby a complete loss of actin filaments, resulting in rounding of cultured monolayer cells (1). The ADPribosylation at Arg-177 turns G-actin monomers into "cappin...

The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.

Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms.

SummaryClostridium botulinum C2 toxin is an ADPribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft-dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant-negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA-mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin-independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho-mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamindependent mechanisms and we provide evidence for involvement of clathrin and Rho.